Neuregulin based methods and compositions for treating cardiovascular diseases

ABSTRACT

The present invention relates to compositions and methods for preventing, treating or delaying various cardiovascular diseases or disorders in mammals, particularly in humans. More particularly, the present invention provides for compositions and methods for preventing, treating or delaying various cardiovascular diseases or disorders using, inter alia, a neuregulin protein, or a functional fragment thereof, or a nucleic acid encoding a neuregulin protein, or a functional fragment thereof, or an agent that enhances production and/or function of said neuregulin.

The present application is related to PCT/CN02/00349, filed May 24,2002, PCT/CN02/00664, filed Sep. 18, 2002, Chinese Patent ApplicationNo. 02145145.1, filed Nov. 8, 2002 and Chinese Patent Application No.03109976.9, filed Apr. 9, 2003. The disclosures of the aboveapplications are incorporated by reference in their entireties.

TECHNICAL FIELD

The present invention relates to compositions and methods forpreventing, treating or delaying various cardiovascular diseases ordisorders in mammals, particularly in humans. More particularly, thepresent invention provides for compositions and methods for preventing,treating or delaying various cardiovascular diseases or disorders using,inter alia, a neuregulin protein, or a functional fragment thereof, or anucleic acid encoding a neuregulin protein, or a functional fragmentthereof, or an agent that enhances production and/or function of saidneuregulin.

BACKGROUND ART

Viral myocarditis is inflammation of heart muscle caused by orassociated with various viruses, which induce focal or diffuseinflammatory exudation in interstitial myocardium and degeneration,necrosis or lysis of cardiac muscle fiber. Viral myocarditis may becomplicated by pericarditis or endocarditis. The outcome of viralmyocarditis includes myocardial damage, heart dysfunction, arrhythmiaand systemic symptoms. Viral myocarditis occurs at any age. In recentyears, the incidence of viral myocarditis is increasing. The sequela ofacute viral myocarditis include arrhythmia, heart failure, cardiacshock, and even sudden death. The disease may be prolonged with cardiachypertrophy and permanent cardiac muscle injury. Cardiomyopathy finallyresults from immune reaction. The treatment of viral myocarditisincludes antibiotics, heart protective agents, antioxidant such as highdose vitamin C, vitamin E, coenzyme Q10, nutrients for myocardium suchas energy combination to provide enough nutrition for myocardium. Thesemeasures are applied to improve heart function, repair cardiac damage,and prevent heart failure, but the efficacy is not so ideal, especiallyfor the structural and functional changes in myocarditis. No reliabletherapy is available for these pathological changes.

Dilated (congestive) cardiomyopathy (DCM) is the eventual outcome ofvarious myocardial diseases of unidentified origin. Its pathologicalchanges are non-rheumatic, non-hypertensive and not due to coronaryheart disease. The main clinical features of dilated cardiomyopathy isventricular hypertrophy, myocardial pump function failure, or congestiveheart failure. Hypertrophy of nucleus and organelles is generally seenin mild myocardium damage of dilated cardiomyopathy. Structural change,cell death and the resultant fibrosis is usually seen in severe damage.The etiology and pathogenesis of DCM remains to be clarified. Theprognosis of DCM is very poor because no effective therapy is available.Most of such patients die of increasingly worsen heart failure. Suddendeath may result from arrhythmia. At present, no specific effectivetherapy is available for DCM. The routine treatment for DCM includesadministration of cardiac tonics, diuretics, ACEI and other drugs. Inaddition, third generation calcium antagonist amlodipine and thirdgeneration β-receptor antagonist such as carvedilol are also used totreat DCM. It is controversial about the effect of thyroxin and growthhormone because no randomized double-blind clinical trial evidence isavailable. Volume reduction surgery of left ventricle can decrease theinside diameter of left ventricle and improve cardiac functiontemporarily. However, the higher mortality associated withpost-operational heart failure and arrhythmia hampers the application ofthis method. Dynamic cardiomyoplasty is useful in improving cardiacfunction, but patient can not tolerate the relatively extensive trauma.Therefore, this operation is only used as alternative to hearttransplant. In surgery, heart transplant is radical treatment for DCM.However, lack of donor heart, expensive cost, post-operation infectionand transplant rejection are the main obstacles. Therefore, new therapyfor DCM is urgently needed in clinical practice.

Doxorubicin or adriamycin (ADM) belongs to anthracycline anticanceragents with a broad spectrum and potent anti-tumor activity. It has beenwidely used in clinical practice to treat various malignant tumors suchas lung cancer, breast cancer, bladder carcinoma, testis carcinoma,thyroid cancer, soft tissue carcinoma, osteosarcoma, neuroblastoma,acute leukemia, malignant lymphoma, gastric carcinoma, liver cancer,esophageal carcinoma, and cervical carcinoma. ADM acts throughincorporation with DNA to inhibit DNA synthesis. The inhibition effectis observed in S, M, G1 and G2 phases, but the activity is most activein S phase. It has been discovered that a higher dose of chemotherapyagents produces better clinical efficacy and longer survival in unittime for breast cancer patients, whether metastatic or post-operativechemotherapy. Other studies have confirmed the importance of higher doseand dose potency, as well as the relation between dose, dose potency andefficacy.

However, the application of ADM is restricted by its toxic side effects.The toxicity of ADM includes bone marrow depression. In about 60-80%patients, leukocyte and platelet counts decrease to the lowest level10-15 days after administration, and recover to normal levels 21 dayslater. Major digestive tract reaction are nausea, vomiting, anorexia,gastritis and even ulcer and stomatitis. Alopecia also occurs in nearlyall patients treated with ADM, although this effect can be reversedafter withdrawal.

More importantly, the major factor preventing clinical application ofhigher dose ADM and other anthracycline agents is cardiac toxicity.Cardiac toxicity of ADM may result from the production of oxygen-derivedfree radicals. The semiquinone group in ADM can induce an oxidoreductionreaction, which leads to enhanced lipid peroxidation, contributing tocellular damage. The free radicals damage cell membrane and organellemembrane, and modify the function of membrane proteins and enzymeactivity. These changes can lead to intracellular calcium overload,inhibition of DNA and protein synthesis, and energy metabolism disorder,which inevitably harm the cardiac contraction and relaxation process.

Cardiac toxicity occurs when ADM accumulates in the body because ADM hasa higher affinity for cardiac tissue than for other tissues. Thus, theheart is more vulnerable to the toxic effect of ADM. Cardiac toxicitymay be acute or chronic. Clinical features of acute cardiac toxicityinclude cardiac functional changes, such as sinus tachycardia,arrhythmia, conduction block, ST-T segment alteration, etc. Variousarrhythmia may be present at early stages of ADM therapy. Acute cardiactoxicity can also include decrease of the left ventricular ejectionfraction (LVEF), as well as decreases in stroke volume (SV), cardiacoutput (CO) and cardiac index (CI). ADM has also been suggested toinhibit the contraction and pumping ability of the left ventricle.Chronic cardiac toxicity include irreversible congestive heart failure.Once a person suffers from congestive heart failure, the mortalitydecreases to about 30% to 50%. Because of the cardiac toxicityassociated with ADM, some patients have to terminate ADM therapy, orreduce the dosage or length of ADM use, affecting the efficacy of ADMtherapy.

Studies have been conducted to find ways to reduce the toxicity of ADM,while maintaining the therapeutic effect. Certain measures, such asdecreasing the total dose of ADM or administering myocardial nutrientssuch as a high dose of vitamin C, may be helpful in protectingmyocardium. Regular blood transfusion and administering an ironchelating agent ICRF-187 also have some effect in protecting myocardium.However, although these supportive agents are beneficial to the cardiacmuscle, they have little effect on the cardiac toxicity induced by ADM.Thus, there exists a need in the art for preventing, treating ordelaying the cardiac toxicity of ADM without affecting its efficacy.

Heart failure is caused by many etiology such as coronaryarteriosclerosis, hypertension and inflammation induced myocardialinjury, finally leading to refractory heart disease. These factors causechanges in myocardial cell structure and function, eventually resultingin lower ventricular pumping function and heart failure. The incidenceand mortality of heart failure is very high worldwide, being one of themost severe lethal diseases. In the United States, 30%-40% of thecongestive heart failure (CHF) lead to hospitalization annually.Mortality 5 years after establishment of the diagnosis of CHF is60%(male) and 45%(female). Mean survival time is 3.2 years (male) and5.4 years (female), while the survival rate of patients with final stageCHF is only about 20%.

Neuregulin-1 (NRG-1), also named as Neu Differentiation Factor (NDF) orGlia1 Growth Factor (GGF), is a ligand of ErbB3 and ErbB4. The study inneuregulin gene defective mouse fetus demonstrates that neuregulin isessential for the development of heart and nervous system. However, thedata are limited about the way neuregulin controls cell differentiationand downstream signal transduction. At early stage of heart development,neuregulin and ErbB receptor are expressed in the lining of endocardiumand cardiac myocytes respectively. As these 2 layers are separatedwidely, neuregulin has to pass through the space between these 2 layersbefore it can activate ErbB receptor. The activation of ErbB receptorsin cardiocyte is helpful for cardiocyte and its migration intoendocardium. WO 00/37095 shows that neuregulin can enhance thedifferentiation of cardiac myocytes, strengthen the combination ofsarcomere and cytoskeleton, as well as intercellular cohesion. WO00/37095 also shows that neuregulin can be used to detect, diagnose andtreat heart diseases. WO 00/37095 further shows that neuregulin and itsanalogues can promote cardiocyte differentiation in vitro, inducereconstruction of sarcomere and cytoskeleton in cardiocyte andintercellular cohesion, identify the polypeptide or compound that caninhibit neuregulin. The identified polypeptide or compound can be usedto treat heart diseases and heart failure.

There exists a need in the art for more efficient and/or cost effectiveneuregulin related treatments for viral myocarditis, dilatedcardiomyopathy, the cardiac toxicity of a prophylactic or therapeuticagent, e.g., ADM, without affecting its efficacy and myocardialinfarction. The present invention addresses these and other relatedneeds in the art.

DISCLOSURE OF THE INVENTION

Treating Viral Myocarditis or Dilated (Congestive) Cardiomyopathy (DCM)

In one aspect, the present invention is directed to a method forpreventing, treating or delaying viral myocarditis or dilated(congestive) cardiomyopathy (DCM) in a mammal, which method comprisesadministering to a mammal, to which such prevention, treatment or delayis needed or desirable, an effective amount of a neuregulin protein, ora functional fragment thereof, or a nucleic acid encoding a neuregulinprotein, or a functional fragment thereof, or an agent that enhancesproduction and/or function of said neuregulin, whereby said viralmyocarditis or DCM is prevented, treated or delayed. Preferably, theneuregulin protein, or a functional fragment thereof, or a nucleic acidencoding a neuregulin protein, or a functional fragment thereof, or anagent that enhances production and/or function of said neuregulin isadministered in vivo.

In another aspect, the present invention is directed to a combination,which combination comprises an effective amount of a neuregulin protein,or a functional fragment thereof, or a nucleic acid encoding aneuregulin protein, or a functional fragment thereof, or an agent thatenhances production and/or function of said neuregulin, and an effectiveamount of a prophylactic or therapeutic agent for viral myocarditis ordilated (congestive) cardiomyopathy (DCM).

In still another aspect, the present invention is directed to apharmaceutical composition for preventing, treating or delaying viralmyocarditis or dilated (congestive) cardiomyopathy (DCM) in a mammal,which pharmaceutical composition comprises an effective amount of aneuregulin protein, or a functional fragment thereof, or a nucleic acidencoding a neuregulin protein, or a functional fragment thereof, or anagent that enhances production and/or function of said neuregulin.

Treating Cardiac Toxicity Associated with Prophylactic or TherapeuticAgents

It is an object of the present invention to provide methods forpreventing, treating or delaying cardiac toxicity associated withprophylactic or therapeutic agents. In particular, it is an object ofthe present invention to provide methods for preventing, treating ordecreasing cardiac toxicity of drug-induced cardiomyopathy.

The present invention provides methods for preventing, treating ordelaying cardiac toxicity in a mammal to which such prevention,treatment or delay is needed or desirable, comprising administering to amammal in vivo an effective amount of a prophylactic or a therapeuticagent and an effective amount of: (i) a neuregulin protein or afunctional fragment thereof; (ii) a nucleic acid encoding a neuregulinprotein or a functional fragment thereof; or (iii) an agent thatenhances production or function of said neuregulin, whereby the cardiactoxicity associated with administration of said prophylactic ortherapeutic agent is prevented, treated or delayed.

The present invention can be used to prevent, treat or delay anyclinical manifestations of cardiac toxicity known to one of ordinaryskill in the art, including but not limited to acute or chronic cardiactoxicity. For example, the present invention can be used to prevent,treat or delay tachycardia, arrhythmia and congestive heart failure. Ina particular embodiment, the present invention can be used to prevent,treat or delay clinical manifestations of acute cardiac toxicity such assinus tachycardia, arrhythmia, conduction block and ST-T segmentalteration, as well as decreases in the left ventricular ejectionfraction (LVEF), stroke volume (SV), cardiac output (CO) or cardiacindex (CI). The present invention can also be used to prevent, treat ordelay cardiac toxicity comprising the inhibition of the contracting andpumping ability of the left ventricle.

The present methods can be used to prevent, treat or delay cardiactoxicity associated with any prophylactic or therapeutic agent. In oneembodiment, the prophylactic or therapeutic agent producesoxygen-derived free radicals, which cause cardiac toxicity. In anotherembodiment, the prophylactic or therapeutic agent enhances lipidperoxidation, which causes cardiac toxicity.

The present methods can be used to prevent, reduce or delay cardiactoxicity associated with anti-neoplasm agents. The anti-neoplasm agentis preferably an anthracycline. In a specific embodiment, theanti-neoplasm agent has the following formula I:

wherein R1 is methoxy or hydrogen; and R2, R3 and R4 are hydroxy orhydrogen. Non-limiting examples of anthracycline for use in the presentmethods include adriamycin (or doxorubicin), daunorubicin, epirubicin,idarubicin, mitoxantrone, mitomycin, bleomycin, cyclophosphamide,fluorouracil, actinomycin D and vincristine. In one aspect, the presentinvention is directed to the use of neuregulin as an anti-cardiotoxicagent for preventing, treating or delaying cardiac toxicity induced bychemotherapeutic agents such as adriamycin (ADM), alone or incombination with another reagent.

The present methods can be used to prevent, reduce or delay cardiactoxicity associated with antipsychotic agents. The antipsychotic agentcan be chlorpromazine, perphenazine or trifluperazine.

The present methods can be used to prevent, reduce or delay cardiactoxicity associated with tricyclic antidepressants. The tricyclicantidepressant can be chlorimipramine, amitriptyline or doxepin.

The present methods can be used to prevent, reduce or delay cardiactoxicity associated with interferon, e.g., interferon-α, or interleukin,e.g., interleukin-2.

The present methods can be used to prevent, reduce or delay cardiactoxicity associated with anti-infectious agents, e.g., emetine.

The neuregulin agent for use in the present methods can be neuregulin 1,neuregulin 2, neuregulin 3, or neuregulin 4. In a particular embodiment,the neuregulin for use in the present methods is neuregulin α2 orneuregulin β2. In another embodiment, the neuregulin fragment is aneuregulin β2 comprising an amino acid sequence set forth in SEQ IDNO:4.

The neuregulin agent can be administered as a protein or a functionalfragment thereof. The neuregulin agent can also be administered as anucleic acid encoding a neuregulin protein or a functional fragmentthereof. Any agent that enhances production or function of neuregulincan also be administered. The neuregulin agent can be administered aloneor with a pharmaceutically acceptable carrier or excipient. Theneuregulin protein or a functional fragment thereof, or a nucleic acidencoding a neuregulin protein or a functional fragment thereof, or anagent that enhances production or function of said neuregulin, can beadministered prior to, concurrently with, or subsequent to theadministration of the prophylactic or therapeutic agent.

In one embodiment, the prophylactic or therapeutic agent is administeredin an amount that is higher than a maximally allowed amount when theprophylactic or therapeutic agent is administered in the absence of theneuregulin protein or a functional fragment thereof, or a nucleic acidencoding a neuregulin protein or a functional fragment thereof, or anagent that enhances production or function of said neuregulin.

In a particular embodiment, the present invention provides methods forpreventing, treating or delaying cardiac toxicity in a human to whichsuch prevention, treatment or delay is needed or desirable. Preferably,the human has a malignant tumor such lung cancer, breast cancer, bladdercarcinoma, testis carcinoma, thyroid cancer, soft tissue carcinoma,osteosarcoma, neuroblastoma, acute leukemia, malignant lymphoma, gastriccarcinoma, liver cancer, esophageal carcinoma, or cervical carcinoma.

Treating Myocardial Infarction

In one aspect, the present invention is directed to a combination, whichcombination comprises an effective amount of a neuregulin protein, or afunctional fragment thereof, or a nucleic acid encoding a neuregulinprotein, or a functional fragment thereof, or an agent that enhancesproduction and/or function of said neuregulin, and an effective amountof a prophylactic or therapeutic agent for myocardial infarction.

In another aspect, the present invention is directed to a kit, which kitcomprises an above-described combination in a container and aninstruction for using said combination in preventing, treating ordelaying myocardial infarction.

In still another aspect, the present invention is directed to a methodfor preventing, treating or delaying myocardial infarction in a mammal,which method comprises administering to a mammal, to which suchprevention, treatment or delay is needed or desirable, an effectiveamount of a neuregulin protein, or a functional fragment thereof, or anucleic acid encoding a neuregulin protein, or a functional fragmentthereof, or an agent that enhances production and/or function of saidneuregulin, whereby said myocardial infarction is prevented, treated ordelayed.

Pharmaceutical Compositions

In one aspect, the present invention is directed to a pharmaceuticalcomposition for preventing, treating or delaying a disease in a mammal,which composition comprises a neuregulin protein, or a functionalfragment thereof: a) in a safety dosage equals to or less than about 170U/kg; or b) in a total regimen equals to or less than about 3,600 U/kg.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates construction of an engineered bacterial strain forrecombinantly producing a neuregulin protein.

FIG. 2 depicts PCR amplification of human neuregulin gene. Lane 1: 183bp neuregulin gene obtained by RT-PCR; and Lanes 2 and 3: DNA markers.

FIG. 3 depicts physical map of plasmid PET22b.

FIG. 4 depicts identification of recombinant plasmid by endonucleasedigestion. Lanes 1 and 3: DNA marker; and lane 2: fragments after enzymedigestion.

FIG. 5 depicts screening for expression of an engineered strain. Lane 1:Marker; lane 2: engineered strain without induction; lane 3: 1 hourafter induction; lane 4: 2 hours after induction; lane 5: 3 hours afterinduction; lane 6: 4 hours after induction; and lanes 7-9: differentstrains with induction.

FIG. 6 depicts pathologic section of myocardial tissue inpseudo-operation group rat (1)(10×10), showing that there was nospecific pathological change.

FIG. 7 depicts large area of red stained region in the model group(10×10).

FIG. 8 illustrates sporadic distribution of red stained myocardial cellsof the rhNRG-1 β group (20 μg/kg) (I) (10×10).

FIG. 9 illustrates patchy distribution of red stained myocardial cellsand connective tissue of the medium dosage (10 μg/kg) of rhNRG-1 β group(I) (10×10).

FIG. 10 illustrates patchy distribution of red stained myocardial cellsand connective tissue of the low dosage (5 μg/kg) of rhNRG-1 β group (I)(10×10).

FIG. 11 depicts pathohistologic section of myocardial tissue inpseudo-operation group (II) (10×10), showing that there was no specificpathologic changes of the myocardial tissue.

FIG. 12 depicts large area of red stained region was seen in the modelgroup (II) (10×10).

FIG. 13 depicts sporadic distribution of red stained myocardial cellsseen in high dosage of rhNRG-1 β (20 μg/kg) group (II) (10×10).

FIG. 14 depicts patchy distribution of red stained myocardial cells andfiber tissue seen in medium dosage level of rhNRG-1 β S177-Q237 (10μs/kg) group (II) (10×10).

FIG. 15 depicts patchy distribution of red stained myocardial cells andfiber tissue seen in low dosage level of rhNRG-1 β S177-Q237 (5 μg/kg)group (II) (10×10).

FIG. 16 illustrates effect of rhNRG-1 β on capillary regeneration inmyocardial fibrotic area of model animal (I) (HE stained, 50×); A:Pseudo-operation group: normal myocardial structure, with fibroticchanges; B: Model animal group: marked myocardial fibrosis and smallamount of capillary proliferation was seen; C: 20 μg/kg rhNRG-1 β group:patchy fibrotic changes in myocardium, significant capillaryproliferation; D: 10 μg/kg rhNRG-1 β group: marked fibrotic changes withrelatively large number of capillary proliferation; and E: 5 μg/kgrhNRG-1 β group: patchy fibrotic changes and capillary proliferationcould be seen.

FIG. 17 illustrates effect of rhNRG-1 β on capillary proliferation inmyocardial fibrotic area of model animal (II) (HE stained, 50×); A:Pseudo-operation group: normal myocardial structure, with fibroticchanges; B: Model animal group: marked myocardial fibrotic changes,small number of capillary proliferation could be seen; C: 20 μg/kgrhNRG-1 β group: patchy fibrotic changes in myocardium, significantcapillary proliferation; D: 10 μg/kg rhNRG-1 β group: marked fibroticchanges, there were relatively large number of capillary proliferation;and E: 5 μg/kg rhNRG-1 β group: patchy fibrotic changes, capillaryproliferation could be seen.

FIG. 18 illustrates myocardium pathologic section of SD rat toxicmyocarditis induced by Adriamycin; a: normal control group: pathologicscore of myocardium was 0, without myocardial cells atrophy andhypertrophy, with vacuole formation, cross striation can be clearlyseen; myocardium arranged regularly; no abnormality of endocardium andpericardium; no changes of the vessels and interstitial tissue; b: modelanimal group: pathologic score of the myocardium was 3, large area ofmyocardial cell necrosis and dissolution.

FIG. 19 illustrates effect of rhNRG-1 β on survival rate of the modelanimals

FIG. 20 illustrates effect of rhNRG-1 β on myocardial structure of modelanimals.

FIG. 21 illustrates effect of rhNRG-1 β S177-Q237 on myocardialpathologic damage in mice infected by virus (I); A: Normal group; B:Model group; C: Placebo control group; D: High dosage level group; E:Medium dosage level group; and F: Low dosage level group.

FIG. 22 illustrates effect of rhNRG-1 β S177-Q237 on myocardialpathologic damage in mice infected by virus; A: Normal group; B: Modelgroup; C: Placebo control group; D: High dosage level group; E: Mediumdosage level group; and F Low dosage level group.

FIG. 23 illustrates effect of rhNRG-1 β S177-Q237 administered fordifferent number of days on myocardial pathologic changes in miceinfected by virus (I) (HE stained, 40×); A: Normal control group; B:model group; C: rhNRG-1 β S177-Q237 for 3-day group; D: rhNRG-1 βS177-Q237 for 5-day group; and E: rhNRG-1 β S177-Q237 for 7-day group.

FIG. 24 illustrates effect of rhNRG-1 β administered for differentnumber of days on myocardial pathologic changes in mice infected byvirus (II) (HE stained, 40×); A: Normal control group; B: model group;C: rhNRG-1 β S177-Q237 for 3-day group; D: rhNRG-1 β for 5-day group;and E: rhNRG-1 β S177-Q237 for 7-day group.

MODES OF CARRYING OUT THE INVENTION

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the subsections thatfollow.

A. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this invention belongs. All patents, applications,published applications and other publications referred to herein areincorporated by reference in their entirety. If a definition set forthin this section is contrary to or otherwise inconsistent with adefinition set forth in the patents, applications, publishedapplications and other publications that are herein incorporated byreference, the definition set forth in this section prevails over thedefinition that is incorporated herein by reference.

As used herein, “a” or “an” means “at least one” or “one or more.”

As used herein, “neuregulin” refers to proteins or peptides that canactivate ErbB2/ErbB4 or ErbB2/ErbB3 heterodimers protein kinases, suchas all neuregulin isoforms, neuregulin EGF domain alone, neuregulinmutants, and any kind of neuregulin-like gene products that alsoactivate the above receptors. An exemplary neuregulin fragment is apolypeptide fragment of human neuregulin β 2 isomer, which containsreceptor-binding domain, i.e., EGF-like domain. This polypeptide canactivate ErbB receptor of EGF receptor family and modulate itsbiological reactions, e.g., stimulate breast cancer cell differentiationand milk protein secretion; induce the differentiation of neural crestcell into Schwann cell; stimulate acetylcholine synthesis in skeletalmuscle cell; and improve cardiocyte survival and DNA synthesis.Neuregulin also includes those variants with conservative amino acidsubstitutions that do not substantially alter their its anti-viralmyocarditis, anti-DCM, anti-cardiotoxic, or anti-myocardial infarctionactivity. Suitable conservative substitutions of amino acids are knownto those of skill in this art and may be made generally without alteringthe biological activity of the resulting molecule. Those of skill inthis art recognize that, in general, single amino acid substitutions innon-essential regions of a polypeptide do not substantially alterbiological activity (see, e.g., Watson et al. Molecular Biology of theGene, 4th Edition, 1987, The Bejaemin/Cummings Pub. co., p. 224).Neuregulin protein encompasses a neuregulin protein and peptide.Neuregulin nucleic acid encompasses neuregulin nucleic acid andneuregulin oligonucleotide.

As used herein, “epidermal growth factor-like domain” or “EGF-likedomain” refers to a polypeptide motif encoded by the neuregulin genethat binds to and activates ErbB2, ErbB3, ErbB4, or combinationsthereof, and bears a structural similarity to the EGF receptor-bindingdomain as disclosed in WO 00/64400, Holmes et al., Science,256:1205-1210 (1992); U.S. Pat. Nos. 5,530,109 and 5,716,930; Hijazi etal., Int. J. Oncol., 13:1061-1067 (1998); Chang et al., Nature,387:509-512 (1997); Carraway et al., Nature, 387:512-516 (1997);Higashiyama et al., J. Biochem., 122:675-680 (1997); and WO 97/09425.

As used herein, a “functional derivative or fragment” of neuregulinrefers to a derivative or fragment of the neuregulin protein or itsencoding nucleic acid that still substantially retains its anti-viralmyocarditis, anti-DCM, anti-cardiotoxic, or anti-myocardial infarctionactivity. Normally, the derivative or fragment retains at least 50% ofits anti-viral myocarditis, anti-DCM, anti-cardiotoxic, oranti-myocardial infarction activity. Preferably, the derivative orfragment retains at least 60%, 70%, 80%, 90%, 95%, 99% and 100% of itsanti-viral myocarditis, anti-DCM, anti-cardiotoxic, or anti-myocardialinfarction activity.

As used herein, an “agent that enhances production of neuregulin” refersto a substance that increases transcription and/or translation of aneuregulin gene, or a substance that increases post-translationalmodification and/or cellular trafficking of a neuregulin precursor, or asubstance that prolongs half-life of a neuregulin protein.

As used herein, an “agent that enhances function of neuregulin” refersto a substance that increases potency of the neuregulin's anti-viralmyocarditis, anti-DCM activity, anti-cardiotoxic activity, oranti-myocardial infarction activity, or a substance that increasessensitivity of a neuregulin's natural ligand in an anti-viralmyocarditis, anti-DCM, anti-cardiotoxic activity, or anti-myocardialinfarction signaling pathway, or a substance that decreases potency of aneuregulin's antagonist. Such an agent is not a neuregulin protein orits encoding nucleic acid.

As used herein, a “combination” refers to any association between two oramong more items.

As used herein, a “composition” refers to any mixture of two or moreproducts or compounds. It may be a solution, a suspension, liquid,powder, a paste, aqueous, non-aqueous or any combination thereof.

As used herein, “erb” refers to two oncogenes, erb A and erb B,associated with erythroblastosis virus (an acute transformingretrovirus).

As used herein, “an effective amount of a compound for treating aparticular disease” is an amount that is sufficient to ameliorate, or insome manner reduce the symptoms associated with the disease. Such amountmay be administered as a single dosage or may be administered accordingto a regimen, whereby it is effective. The amount may cure the diseasebut, typically, is administered in order to ameliorate the symptoms ofthe disease. Repeated administration may be required to achieve thedesired amelioration of symptoms.

As used herein, “heart failure” is meant an abnormality of cardiacfunction where the heart does not pump blood at the rate needed for therequirements of metabolizing tissues. Heart failure includes a widerange of disease states such as congestive heart failure, myocardialinfarction, tachyarrythmia, familial hypertrophic cardiomyopathy,ischaemic heart disease, idiopathic dilated cardiomyopathy, andmyocarditis. The heart failure can be caused by any number of factors,including ischaemic, congenital, rheumatic, or idiopathic forms. Chroniccardiac hypertrophy is a significantly diseased state which is aprecursor to congestive heart failure and cardiac arrest.

As used herein, “production by recombinant means” refers to productionmethods that use recombinant nucleic acid methods that rely on wellknown methods of molecular biology for expressing proteins encoded bycloned nucleic acids.

As used herein, “complementary” when referring to two nucleic acidmolecules, means that the two sequences of nucleotides are capable ofhybridizing, preferably with less than 25%, more preferably with lessthan 15%, even more preferably with less than 5%, most preferably withno mismatches between opposed nucleotides. Preferably the two moleculeswill hybridize under conditions of high stringency.

As used herein: “stringency of hybridization” in determining percentagemismatch is as follows:

1) high stringency: 0.1×SSPE, 0.1% SDS, 65° C.;

2) medium stringency: 0.2×SSPE, 0.1% SDS, 50° C. (also referred to asmoderate stringency); and

3) low stringency: 1.0×SSPE, 0.1% SDS, 50° C.

It is understood that equivalent stringencies may be achieved usingalternative buffers, salts and temperatures.

As used herein, “vector (or plasmid)” refers to discrete elements thatare used to introduce heterologous DNA into cells for either expressionor replication thereof. Selection and use of such vehicles are wellknown within the skill of the artisan. An expression vector includesvectors capable of expressing DNA's that are operatively linked withregulatory sequences, such as promoter regions, that are capable ofeffecting expression of such DNA fragments. Thus, an expression vectorrefers to a recombinant DNA or RNA construct, such as a plasmid, aphage, recombinant virus or other vector that, upon introduction into anappropriate host cell, results in expression of the cloned DNA.Appropriate expression vectors are well known to those of skill in theart and include those that are replicable in eucaryotic cells and/orprokaryotic cells and those that remain episomal or those whichintegrate into the host cell genome.

As used herein, “a promoter region or promoter element” refers to asegment of DNA or RNA that controls transcription of the DNA or RNA towhich it is operatively linked. The promoter region includes specificsequences that are sufficient for RNA polymerase recognition, bindingand transcription initiation. This portion of the promoter region isreferred to as the promoter. In addition, the promoter region includessequences that modulate this recognition, binding and transcriptioninitiation activity of RNA polymerase. These sequences may be cis actingor may be responsive to trans acting factors. Promoters, depending uponthe nature of the regulation, may be constitutive or regulated.Exemplary promoters contemplated for use in prokaryotes include thebacteriophage T7 and T3 promoters, and the like.

As used herein, “operatively linked or operationally associated” refersto the functional relationship of DNA with regulatory and effectorsequences of nucleotides, such as promoters, enhancers, transcriptionaland translational stop sites, and other signal sequences. For example,operative linkage of DNA to a promoter refers to the physical andfunctional relationship between the DNA and the promoter such that thetranscription of such DNA is initiated from the promoter by an RNApolymerase that specifically recognizes, binds to and transcribes theDNA. In order to optimize expression and/or in vitro transcription, itmay be necessary to remove, add or alter 5′ untranslated portions of theclones to eliminate extra, potential inappropriate alternativetranslation initiation (i.e., start) codons or other sequences that mayinterfere with or reduce expression, either at the level oftranscription or translation. Alternatively, consensus ribosome bindingsites (see, e.g., Kozak, J. Biol. Chem., 266:19867-19870 (1991)) can beinserted immediately 5′ of the start codon and may enhance expression.The desirability of (or need for) such modification may be empiricallydetermined.

As used herein, “amelioration” of the symptoms of a particular disorderby administration of a particular pharmaceutical composition refers toany lessening, whether permanent or temporary, lasting or transient thatcan be attributed to or associated with administration of thecomposition.

As used herein, “a therapeutic agent” refers to any conventional drug ordrug therapies which are known to those skilled in the art, including,but not limited to prophylactic or chemotherapeutic agents.

As used herein, “therapeutically effective amount” refers to that amountthat is sufficient to ameliorate, or in some manner reduce the symptomsassociated with a disease. Such amount may be administered as a singledosage or according to a regimen. Repeated administration may berequired to achieve the desired amelioration of symptoms.

As used herein, “administration” or “administering” a compound refers toany suitable method of providing a compound to a subject.

As used herein, “treatment” or “treating” refer to any manner in whichthe symptoms of a condition, disorder or disease are ameliorated orotherwise beneficially altered. Treatment also encompasses anypharmaceutical use of the compositions herein. Amelioration of symptomsof a particular disorder refers to any lessening of symptoms, whetherpermanent or temporary, that can be attributed to or associated withadministration of the composition.

As used herein, “neoplasm (neoplasia)” refers to abnormal new growth ortumor growth, which may be benign or malignant. Unlike hyperplasia,neoplastic proliferation persists even in the absence of the originalstimulus.

As used herein, “an anti-neoplasm agent” refers to any agents used toprevent the occurrence or lessen the severity of neoplasm, tumor orcancer. These include, but are not limited to, anti-angiogenic agents,alkylating agents, antimetabolites, natural products, platinumcoordination complexes, anthracenedione, substituted urea,methylhydrazine derivatives, adrenocortical suppressants, hormones,antagonists, oncogene inhibitors, tumor suppressor genes or proteins,anti-oncogene antibodies, or anti-oncogene antisense oligonucleotides.

As used herein, “anti-psychotic agent” refers to any agents used in thetreatment of psychiatric disorders. These include, but are not limitedto, tricyclic phenothiazines, thioxanthenes, and dibenzepines, as wellas butyrophenones and congeners, other heterocyclics, and experimentalbenzamides.

As used herein, “tricyclic antidepressants” refers to any agents used inthe treatment of depression. These include, but are not limited to,agents that inhibit norepinephrine and serotonin uptake into nerveendings, and thus leading to sustained facilitation of noradrenergicfunction in the brain.

As used herein, “anti-infectious agent” refers to any agents used in thetreatment of infectious diseases. These include, but are not limited to,agents for use against parasitic infections, bacterial and microbialinfections.

As used herein, “safety dosage” refers to a dosage sufficient toprevent, treat or delay a disease in mammal without inducing intolerabletoxic or other side effects in said mammal.

As used herein, “myocardial infarction” refers to blockade of coronaryartery, blood flow interruption, leading to focal necrosis of part ofthe myocardium caused by severe and persistent ischemia.

B. METHODS FOR PREVENTING, TREATING OR DELAYING VIRAL MYOCARDITIS OR DCM

In one aspect, the present invention is directed to a method forpreventing, treating or delaying viral myocarditis or dilated(congestive) cardiomyopathy (DCM) in a mammal, which method comprisesadministering to a mammal, to which such prevention, treatment or delayis needed or desirable, an effective amount of a neuregulin protein, ora functional fragment thereof, or a nucleic acid encoding a neuregulinprotein, or a functional fragment thereof, or an agent that enhancesproduction and/or function of said neuregulin, whereby said viralmyocarditis or DCM is prevented, treated or delayed.

The present method can be used for preventing, treating or delayingviral myocarditis or DCM in any mammals, such as mice, rats, rabbits,cats, dogs, pigs, cows, ox, sheep, goats, horses, monkeys and othernon-human primates. Preferably, the present method is used forpreventing, treating or delaying viral myocarditis or DCM in humans.

Any suitable neuregulin protein, or a functional fragment thereof, or anucleic acid encoding a neuregulin protein, or a functional fragmentthereof, can be used in the present methods. In one specific embodiment,the neuregulin used in the present methods carries out its anti-viralmyocarditis or anti-DCM activity via binding with ErbB2-ErbB4 receptors.In another specific embodiment, neuregulin 1, neuregulin 2, neuregulin 3or neuregulin 4 is used in the present methods. Synonyms of neuregulin 1include heregulin, GGF2 and p185erbB2 ligand. See e.g., WO 00/64400 andU.S. Pat. Nos. 5,530,109 and 5,716,930. Both neuregulin α2 andneuregulin β2 can be used in the present methods. Preferably, aneuregulin β2 fragment comprising an amino acid sequence set forth inSEQ ID NO:4 is used in the present methods.

In still another specific embodiments, neuregulins or functionalfragments thereof disclosed in the following patents, patentapplications and GenBank databases can be used in the present methods:U.S. Pat. Nos. 6,252,051 and 6,121,415 (NRG3); U.S. Pat. No. 6,087,323(neuregulin with p185^(erbB2), p185^(erbB3) or p185^(erbB4) bindingactivity); U.S. Pat. No. 6,033,906 (neuregulin as a ligand for areceptor selected from the group consisting of p185^(erbB2) andp180^(erbB4)); US2002002276 (The chimeric ErbB heteromultimer adhesinsas competitive antagonists or agonists of a neuregulin); WO01/81540(NRG-4); WO01/64877 (NRG1); WO01/64876 (NRG1AG1); WO01/58948(neuregulin-beta); WO01/26607 (SMDF and GGF neuregulin splice variantisoforms); WO00/70322 (CRD-neuregulin); WO00/64400; WO99/18976;WO98/02540 (chimeric ErbB heteromultimer adhesins as competitiveantagonists or agonists of a neuregulin); WO96/30403; WO96/15812;BC017568 (Homo sapiens, Similar to neuregulin 4); BC007675 (Homosapiens, neuregulin 1); AF142632 (Xenopus laevis cysteine-rich domainneuregulin-1); AF194439 (Rattus norvegicus SMDF neuregulin alpha 2a(Nrg1)); AF194438 (Rattus norvegicus SMDF neuregulin beta 1a (Nrg1));HS2NRG12 (Homo sapiens alternatively spliced neuregulin 2 (NRG2));HS2NRG08 (Homo sapiens alternatively spliced neuregulin 2 (NRG2));HS2NRG07 (Homo sapiens alternatively spliced neuregulin 2 (NRG2));AF083067 (Mus musculus neuregulin-4 short isoform (Nrg4)); AF076618(Xenopus laevis neuregulin alpha-1); AF045656 (Gallus gallus neuregulinbeta-2b); AF045655 (Gallus gallus neuregulin beta-2a); AF045654 (Gallusgallus neuregulin beta-1a); MAU96612 (Mesocricetus auratus neuregulin);AF010130 (Mus musculus neuregulin-3 (NRG3)). Preferably, neuregulin(s)disclosed in the GenBank Accession No. NT_(—)007995 (gi:18570363) isused in the present methods.

Any viral myocarditis can be prevented, treated or delayed using thepresent methods. For example, the viral myocarditis caused by orassociated with infection of a Coxsackie Group A virus, Coxsackie GroupB virus, ECHO virus or polio virus can be prevented, treated or delayedusing the present methods. Preferably, the viral myocarditis caused byor associated with infection of a Coxsackie Group B virus is prevented,treated or delayed using the present methods.

Viral myocarditis with various clinical features can be prevented,treated or delayed using the present methods. For example, the viralmyocarditis that is complicated by pericarditis or endocarditis can beprevented, treated or delayed using the present methods. In anotherexample, the viral myocarditis with a clinical feature of myocardialdamage, heart dysfunction, arrhythmia, systemic symptom orcardiomyopathy can be prevented, treated or delayed using the presentmethods. Both the acute and the chronic viral myocarditis can beprevented, treated or delayed using the present methods. Preferably, theacute viral myocarditis having arrhythmia, heart failure or cardiacshock can be prevented, treated or delayed using the present methods.The viral myocarditis that is prolonged with cardiac hypertrophy and/orpermanent cardiac muscle injury may also be prevented, treated ordelayed using the present methods.

DCM with various clinical features can be prevented, treated or delayedusing the present methods. For example, the DCM having ventricularhypertrophy, myocardial pump function failure or congestive heartfailure can be prevented, treated or delayed using the present methods.

The neuregulin protein, or a functional fragment thereof, or a nucleicacid encoding a neuregulin protein, or a functional fragment thereof,can be administered alone. Alternatively, the neuregulin protein, or afunctional fragment thereof, or a nucleic acid encoding a neuregulinprotein, or a functional fragment thereof, can be administered with apharmaceutically acceptable carrier or excipient.

The neuregulin protein, or a functional fragment thereof, or a nucleicacid encoding a neuregulin protein, or a functional fragment thereof,can be administered in vivo, i.e., administered directly into a mammal.Alternatively, the neuregulin protein, or a functional fragment thereof,or a nucleic acid encoding a neuregulin protein, or a functionalfragment thereof, can be administered ex vivo, i.e., administered intocell(s), tissue(s) or organ(s) and such cell(s), tissue(s) or organ(s)carrying the neuregulin protein, or a functional fragment thereof, or anucleic acid encoding a neuregulin protein, or a functional fragmentthereof, can be transferred into a mammal.

In one specific embodiment, a neuregulin protein, or a functionalfragment thereof, is administered. In another specific embodiment, anucleic acid encoding a neuregulin protein, or a functional fragmentthereof, is administered.

The neuregulin protein, or a functional fragment thereof, or a nucleicacid encoding a neuregulin protein, or a functional fragment thereof,can be used alone. Alternatively, the neuregulin protein, or afunctional fragment thereof, or a nucleic acid encoding a neuregulinprotein, or a functional fragment thereof, can be used in combinationwith a prophylactic or therapeutic agent for viral myocarditis or DCM.The neuregulin protein, or a functional fragment thereof, or a nucleicacid encoding a neuregulin protein, or a functional fragment thereof,can be administered prior to, concurrently with, or subsequent to theadministration of the prophylactic or therapeutic agent for viralmyocarditis or DCM.

Any suitable prophylactic or therapeutic agent for viral myocarditis canbe used in the present methods. For example, the prophylactic ortherapeutic agent for viral myocarditis can be an antibiotic, e.g.,penicillin, a heart protective agent, e.g., taurine, an antioxidant,e.g., vitamin C, vitamin E and coenzyme Q10, and a nutrient formyocardium, e.g., an energy combination.

Any suitable prophylactic or therapeutic agent for DCM can be used inthe present methods. For example, the prophylactic or therapeutic agentfor DCM can be a cardiac tonic, e.g., digoxin and cedilanid, a diuretic(Goodman and Gilman's The Pharmacological Basis of Therapeutics (9thEd.), McGraw-Hill (1996) pp 683-713), e.g., an inhibitor of carbonicanhydrase, an osmotic diuretic, an inhibitor of Na⁺—K⁺-2Cl⁻ symport, aninhibitor of Na⁺—Cl⁻ symport, an inhibitor of renal epithelial Na⁺channels and an antagonist of mineralocorticoid receptors, angiotensinI-converting enzyme inhibitor (ACEI), a calcium antagonist, e.g.,amlodipine, and a β-receptor antagonist, e.g., carvedilol.

C. PHARMACEUTICAL COMPOSITIONS, KITS AND COMBINATIONS FOR PREVENTING,TREATING OR DELAYING VIRAL MYOCARDITIS OR DCM

In another aspect, the present invention is directed to a pharmaceuticalcomposition for preventing, treating or delaying viral myocarditis ordilated (congestive) cardiomyopathy (DCM) in a mammal, whichpharmaceutical composition comprises an effective amount of a neuregulinprotein, or a functional fragment thereof, or a nucleic acid encoding aneuregulin protein, or a functional fragment thereof, or an agent thatenhances production and/or function of said neuregulin.

Any suitable neuregulin protein, or a functional fragment thereof, or anucleic acid encoding a neuregulin protein, or a functional fragmentthereof, including the ones described in the above Section B, can beused in the present pharmaceutical compositions. In one specificembodiment, the neuregulin used in the present pharmaceuticalcompositions carries out its anti-viral myocarditis or anti-DCM activityvia binding with ErbB2-ErbB4 receptors. In another specific embodiment,neuregulin 1, neuregulin 2, neuregulin 3 or neuregulin 4 is used in thepresent pharmaceutical compositions. Synonyms of neuregulin 1 includeheregulin, GGF2 and p185erbB2 ligand. See e.g., WO 00/64400 and U.S.Pat. Nos. 5,530,109 and 5,716,930. Both neuregulin α2 and neuregulin β2can be used in the present pharmaceutical compositions. Preferably, aneuregulin β2 fragment comprising an amino acid sequence set forth inSEQ ID NO:4 is used in the present pharmaceutical compositions.

The neuregulin protein, or a functional fragment thereof, or a nucleicacid encoding a neuregulin protein, or a functional fragment thereof,can be used in any suitable dosage ranges. For example, the neuregulinprotein, or a functional fragment thereof, or a nucleic acid encoding aneuregulin protein, or a functional fragment thereof, can have a dosagerange from about 25 μg to about 2,500 μg.

In still another aspect, the present invention is directed to a kit,which kit comprises the above pharmaceutical composition in a containerand an instruction for using said pharmaceutical composition inpreventing, treating or delaying viral myocarditis or DCM.

In yet another aspect, the present invention is directed to acombination, which combination comprises an effective amount of aneuregulin protein, or a functional fragment thereof, or a nucleic acidencoding a neuregulin protein, or a functional fragment thereof, or anagent that enhances production and/or function of said neuregulin, andan effective amount of a prophylactic or therapeutic agent for viralmyocarditis or dilated (congestive) cardiomyopathy (DCM).

Any suitable neuregulin protein, or a functional fragment thereof, or anucleic acid encoding a neuregulin protein, or a functional fragmentthereof, including the ones described in the above Section B, can beused in the present combinations. In one specific embodiment, theneuregulin used in the present combinations carries out its anti-viralmyocarditis or anti-DCM activity via binding with ErbB2-ErbB4 receptors.In another specific embodiment, neuregulin 1, neuregulin 2, neuregulin 3or neuregulin 4 is used in the present combinations. Synonyms ofneuregulin 1 include heregulin, GGF2 and p185erbB2 ligand. See e.g., WO00/64400 and U.S. Pat. Nos. 5,530,109 and 5,716,930. Both neuregulin α2and neuregulin in can be used in the present combinations. Preferably, aneuregulin β2 fragment comprising an amino acid sequence set forth inSEQ ID:1 is used in the present combinations.

Any suitable prophylactic or therapeutic agent for viral myocarditis,including the ones described in the above Section B, can be used in thepresent combinations. For example, the prophylactic or therapeutic agentfor viral myocarditis can be an antibiotic, e.g., penicillin, a heartprotective agent, e.g., taurine, an antioxidant, e.g., vitamin C,vitamin E and coenzyme Q10, and a nutrient for myocardium, e.g., anenergy combination.

Any suitable prophylactic or therapeutic agent for DCM, including theones described in the above Section B, can be used in the presentcombinations. For example, the prophylactic or therapeutic agent for DCMcan be a cardiac tonic, e.g., digoxin and cedilanid, a diuretic, e.g.,an inhibitor of carbonic anhydrase, an osmotic diuretic, an inhibitor ofNa⁺—K⁺-2Cl⁻ symport, an inhibitor of Na⁺—Cl⁻ symport, an inhibitor ofrenal epithelial Na^(±) channels and an antagonist of mineralocorticoidreceptors, angiotensin I-converting enzyme inhibitor (ACEI), a calciumantagonist, e.g., amlodipine, and a β-receptor antagonist, e.g.,carvedilol.

In yet another aspect, the present invention is directed to a kit, whichkit comprises the above combination in a container and an instructionfor using said combination in preventing, treating or delaying viralmyocarditis or DCM.

D. METHODS FOR PREVENTING, TREATING OR DELAYING CARDIAC TOXICITY

Neuregulin has been found to enhance the differentiation of cardiacmyocytes, and strengthen the combination of sarcomere and cytoskeleton,as well as intercellular cohesion (WO 00/37095). Neuregulin can also beused to detect, diagnose and treat heart diseases. In the methods of thepresent invention, neuregulin is used as a cardiocyte protective agentfor preventing, treating or delaying cardiac toxicity due to otherprophylactic or therapeutic agents.

In one aspect, the present invention is directed to a method forpreventing, treating or delaying cardiac toxicity in a mammal to whichsuch prevention, treatment or delay is needed or desirable, comprisingadministering to a mammal in vivo an effective amount of a prophylacticor a therapeutic agent and an effective amount of: (i) a neuregulinprotein or a functional fragment thereof; (ii) a nucleic acid encoding aneuregulin protein or a functional fragment thereof; or (iii) an agentthat enhances production or function of said neuregulin, whereby thecardiac toxicity associated with administration of said prophylactic ortherapeutic agent is prevented, treated or delayed. The present methodcan be used for preventing, treating or delaying cardiac toxicity in anymammals, such as mice, rats, rabbits, cats, dogs, pigs, cows, ox, sheep,goats, horses, monkeys and other non-human primates. Preferably, thepresent method is used for preventing, treating or delaying cardiactoxicity in humans.

1. Neuregulin Agents

Any suitable neuregulin protein or a functional fragment thereof, or anucleic acid encoding a neuregulin protein or a functional fragmentthereof, can be used in the present methods. In one embodiment, themethods of the present invention uses a polypeptide fragment of a humanneuregulin β 2 isomer, which contains the receptor-binding domain (i.e.,an EGF-class region). This polypeptide can activate the erbB receptor ofthe EGF receptor family and modulate its biological reactions (e.g.,stimulate breast cancer cell differentiation and milk protein secretion;induce the differentiation of neural crest cell into Schwann cell;stimulate acetylcholine synthesis in skeletal muscle cell; and/orimprove cardiocyte survival and DNA synthesis). Neuregulin nucleic acidsand proteins can be produced by any suitable methods known in the art,including but not limited to recombinant production, chemical synthesisor a combination of both. Preferably, neuregulin nucleic acids andproteins are produced by recombinant production. (See, Ausubel et al.,Current Protocols in Molecular Biology, John Wiley & Sons, Inc. 2002).

Neuregulin variants with conservative amino acid substitutions that donot substantially alter their anti-cardiotoxic activity can also be usedin the present methods. Suitable conservative substitutions of aminoacids are known to those of skill in this art, and may be made generallywithout altering the biological activity of the resulting molecule.Those of skill in this art recognize that, in general, single amino acidsubstitutions in non-essential regions of a polypeptide do notsubstantially alter biological activity (see e.g., Watson et al.Molecular Biology of the Gene, 4th Edition, page 224, TheBenjamin/Cummings Pub. Co., 1987).

The nucleic acid encoding a neuregulin protein or a functional fragmentthereof, can be used in the form of naked DNA, complexed DNA, cDNA,plasmid DNA, RNA or other mixtures thereof as components of the genedelivery system. In another embodiment, the nucleic acid encoding aneuregulin protein or a functional fragment thereof, is included in aviral vector. Any viral vectors that are suitable for gene therapy canbe used. Non-limiting examples include adenovirus vectors (U.S. Pat. No.5,869,305), simian virus vectors (U.S. Pat. No. 5,962,274),conditionally replicating human immunodeficiency viral vectors (U.S.Pat. No. 5,888,767), retroviruses, SV40, herpes simplex viral ampliconvectors, and vaccinia virus vectors. In addition, the genes can bedelivered in a non-viral vector system such as a liposome wherein thelipid protects the DNA or other biomaterials from oxidation during thecoagulation.

In a specific embodiment, the neuregulin used in the present methodscarries out its anti-cardiotoxic activity via binding with any of theerbB2-erbB4 receptors. In another specific embodiment, neuregulin 1,neuregulin 2, neuregulin 3 or neuregulin 4 is used in the presentmethods. Synonyms of neuregulin 1 include heregulin, GGF2 and p185erbB2ligand. (See e.g., WO 00/64400 and U.S. Pat. Nos. 5,530,109 and5,716,930). Both neuregulin α2 and neuregulin β2 can be used in thepresent methods. Preferably, a neuregulin β2 fragment comprising anamino acid sequence set forth in SEQ ID NO:4 is used in the presentmethods.

In still another specific embodiments, neuregulins or functionalfragments thereof disclosed in the following patents, patentapplications and GenBank databases can be used in the present methods:U.S. Pat. Nos. 6,252,051 and 6,121,415 (NRG3); U.S. Pat. No. 6,087,323(neuregulin with p185^(erbB2), p185^(erbB3) or p185^(erbB4) bindingactivity); U.S. Pat. No. 6,033,906 (neuregulin as a ligand for areceptor selected from the group consisting of p185^(erbB2) andp180^(erbB4)); US2002002276 (chimeric ErbB heteromultimer adhesins ascompetitive antagonists or agonists of a neuregulin); WO01/81540(NRG-4); WO01/64877 (NRG1); WO01/64876 (NRG1AG1); WO01/58948(neuregulin-beta); WO01/26607 (SMDF and GGF neuregulin splice variantisoforms); WO00/70322 (CRD-neuregulin); WO00/64400; WO99/18976;WO98/02540 (chimeric ErbB heteromultimer adhesins as competitiveantagonists or agonists of a neuregulin); WO96/30403; WO96/15812;BC017568 (Homo sapiens, Similar to neuregulin 4); BC007675 (Homosapiens, neuregulin 1); AF142632 (Xenopus laevis cysteine-rich domainneuregulin-1); AF194439 (Rattus norvegicus SMDF neuregulin alpha 2a(Nrg1)); AF194438 (Rattus norvegicus SMDF neuregulin beta 1a (Nrg1));HS2NRG12 (Homo sapiens alternatively spliced neuregulin 2 (NRG2));HS2NRG08 (Homo sapiens alternatively spliced neuregulin 2 (NRG2));HS2NRG07 (Homo sapiens alternatively spliced neuregulin 2 (NRG2));AF083067 (Mus musculus neuregulin-4 short isoform (Nrg4)); AF076618(Xenopus laevis neuregulin alpha-1); AF045656 (Gallus gallus neuregulinbeta-2b); AF045655 (Gallus gallus neuregulin beta-2a); AF045654 (Gallusgallus neuregulin beta-1a); MAU96612 (Mesocricetus auratus neuregulin);AF010130 (Mus musculus neuregulin-3 (NRG3)). Preferably, neuregulin(s)disclosed in the GenBank Accession No. NT_(—)007995 (gi:18570363) isused in the present methods.

2. Prophylactic or Therapeutic Agents

The present invention provides methods for preventing, treating ordelaying cardiac toxicity associated with the administration of aprophylactic or therapeutic agent. The neuregulin agent can beadministered prior to, concurrently with, or subsequent to theadministration of the prophylactic or therapeutic agent. The presentmethods are not limited to preventing, treating or delaying cardiactoxicity associated with specific prophylactic or therapeutic agents.Non-limiting examples of prophylactic or therapeutic agents for use inthe present methods include anti-neoplasm agents, antipsychotic agents,tricyclic depressants, interferons, interleukins, and anti-infectiousagents. Any anti-neoplasm agent can be used in the present methods.Preferably, the anti-neoplasm agent is an anthracyline anti-neoplasmagent having the formula:

wherein R1 is methoxy or hydrogen; and R2, R3 and R4 are hydroxy orhydrogen.

Non-limiting examples of anthracycline anti-neoplasm agent includeadriamycin (or doxorubicin), daunorubicin, epirubicin, idarubicin,mitoxantrone, mitomycin, bleomycin, cyclophosphamide, fluorouracil,actinomycin D, vincristine, and derivatives thereof. (See Goodman &Gilman's, The Pharmacological Basis of Therapeutics, Ninth Edition, pp.1264-1269, McGraw-Hill 1996). Other examples of anti-neoplasm agentsthat can be used in the present methods are described in U.S. PatentApplication No 2002/044919. Other anti-neoplasm agents include, but arenot limited to, cytidine, arabinosyladenine (araC), daunomycin,methotrexate (MTX), fluorinated pyrimidines such as 5-fluorouracil(5-FU), hydroxyurea, 6-mercaptopurine, plant alkaloids such asvincristine (VCR), VP-16 and vinblastine (VLB), alkylating agent,cisplatin, nitrogen Mustard, trisamine, procarbazine, bleomycin,mitomycin C, actinomycin D, or an enzyme such as L-Asparaginase. (SeeGoodman & Gilman's, The Pharmacological Basis of Therapeutics, NinthEdition, pp. 1227-1229).

Any antipsychotic agent can be used in the present methods. Non-limitingexamples of antipsychotic agents include chlorpromazine, perphenazineand trifluperazine. Other examples of antipsychotic agents can be foundin Goodman & Gilman's, The Pharmacological Basis of Therapeutics, NinthEdition, pp. 400-420.

Any tricyclic antidepressant can be used in the present methods.Non-limiting examples of tricyclic antidepressants includechlorimipramine, amitriptyline and doxepin. Other examples ofantipsychotic agents can be found in Goodman & Gilman's, ThePharmacological Basis of Therapeutics, Ninth Edition, pp. 431-434.

Any interferon can be used in the present methods. Preferably, theinterferon is interferon-α. In one embodiment, the interferon is humaninterferon-α. Methods for producing interferon-α can be found in U.S.Pat. Nos. 6,005,075; 5,834,235; 5,503,828; and 4,820,638.

Any interleukin can be used in the present methods. Preferably, theinterleukin is interleukin-2. In one embodiment, the interleukin ishuman interleukin-2. Methods for producing interleukin-2 can be found inU.S. Pat. Nos. 5,834,441; 5,795,777; 5,419,899; and 5,399,699.

Any anti-infectious agent can be used in the present methods.Preferably, the anti-infectious agent is emetine. Other examples ofanti-infectious agent can be found in Goodman & Gilman's, ThePharmacological Basis of Therapeutics, Ninth Edition, pp. 965-1008.

Neuregulin agents can be administered in vivo (i.e., administereddirectly into a mammal). Alternatively, the neuregulin protein or afunctional fragment thereof, or a nucleic acid encoding a neuregulinprotein or a functional fragment thereof, can be administered ex vivo(i.e., administered into cells, tissues or organs, wherein such cells,tissues or organs carrying the neuregulin protein, or a functionalfragment thereof, or a nucleic acid encoding a neuregulin protein, or afunctional fragment thereof, can be transferred into a mammal).

E. TREATING MYOCARDIAL INFARCTION

In one aspect, the present invention is directed to a combination, whichcombination comprises an effective amount of a neuregulin protein, or afunctional fragment thereof, or a nucleic acid encoding a neuregulinprotein, or a functional fragment thereof, or an agent that enhancesproduction and/or function of said neuregulin, and an effective amountof a prophylactic or therapeutic agent for myocardial infarction.Preferably, the present combinations further comprise a pharmaceuticallyacceptable carrier or excipient.

Any suitable neuregulin can be used in the present combinations. Forexample, the neuregulin used in the present combinations can carry outits anti-myocardial infarction activity via binding with ErbB2-ErbB4receptors. In another example, the neuregulin used in the presentcombinations is neuregulin 1, neuregulin 2, neuregulin 3 or neuregulin4. Preferably, the neuregulin 2 is neuregulin α2 or neuregulin f32. Instill another example, the neuregulin used in the present combinationsis a neuregulin fragment comprising an amino acid sequence set forth inSEQ ID NO:4. Any specific neuregulin described in the above sections canalso be used.

Any suitable prophylactic or therapeutic agent can be used in thepresent combinations. For example, the prophylactic or therapeutic agentused in the present combinations can be an angiotensin I-convertingenzyme inhibitor (ACEI), a calcium antagonist, a β-receptor antagonist,aspirin, atropine, nitroglycerin, scopolamine or a thrombolytic agent.

Any suitable ACEI can be used. Exemplary ACEIs include Captopril,Rampril, Lisinopril, Zofenopril and Trandolapril. Any suitable calciumantagonist can be used. Exemplary calcium antagonists include diltiazem.Any suitable β-receptor antagonist can be used. Exemplary β-receptorantagonists include propranolol, metoprolol, atenolol and timolol. Anysuitable thrombolytic agent can be used. Exemplary thrombolytic agentsinclude streptokinase, t-PA and anistreplase.

In another aspect, the present invention is directed to a kit, which kitcomprises an above-described combination in a container and aninstruction for using said combination in preventing, treating ordelaying myocardial infarction.

In still another aspect, the present invention is directed to a methodfor preventing, treating or delaying myocardial infarction in a mammal,which method comprises administering to a mammal, to which suchprevention, treatment or delay is needed or desirable, an effectiveamount of a neuregulin protein, or a functional fragment thereof, or anucleic acid encoding a neuregulin protein, or a functional fragmentthereof, or an agent that enhances production and/or function of saidneuregulin, whereby said myocardial infarction is prevented, treated ordelayed.

Any suitable neuregulin can be used in the present methods. For example,the neuregulin used in the present methods can carry out itsanti-myocardial infarction activity via binding with ErbB2-ErbB4receptors. In another example, the neuregulin used in the presentmethods is neuregulin 1, neuregulin 2, neuregulin 3 or neuregulin 4Preferably, the neuregulin 2 is neuregulin α2 or neuregulin β2. In stillanother example, the neuregulin used in the present methods is aneuregulin β2 fragment comprising an amino acid sequence set forth inSEQ ID NO:4. Any specific neuregulin described in the above sections canalso be used.

In a specific embodiment, the neuregulin protein, or a functionalfragment thereof, the nucleic acid encoding a neuregulin protein, or afunctional fragment thereof, or the agent that enhances productionand/or function of the neuregulin antagonizes the increase of leftventricular end-diastolic (LVEDD) and end-systolic diameters (LVESD)associated with the myocardial infarction. In another specificembodiment, the neuregulin protein, or a functional fragment thereof,the nucleic acid encoding a neuregulin protein, or a functional fragmentthereof, or the agent that enhances production and/or function of theneuregulin antagonizes the decrease of left ventricular EF associatedwith the myocardial infarction.

The present methods can be used to prevent, treat or delay myocardialinfarction in any suitable mammal. Exemplary mammals include mice, rats,rabbits, cats, dogs, pigs, cows, ox, sheep, goats, horses, monkeys andother non-human primates. Preferably, the present methods are used toprevent, treat or delay myocardial infarction in a human.

The present methods can be used to prevent, treat or delay any suitablemyocardial infarction. For example, the present methods can be used toprevent, treat or delay myocardial infarction having a clinical featureselected from the group consisting of left ventricular dilation, reducedsystolic function and increased filling pressure.

The neuregulin protein, or a functional fragment thereof, or a nucleicacid encoding a neuregulin protein, or a functional fragment thereof, orthe agent that enhances production and/or function of the neuregulin,can be administered alone. Preferably, the neuregulin protein, or afunctional fragment thereof, or a nucleic acid encoding a neuregulinprotein, or a functional fragment thereof, or the agent that enhancesproduction and/or function of the neuregulin, is administered with apharmaceutically acceptable carrier or excipient. In a specificembodiment, a neuregulin protein, or a functional fragment thereof, isadministered. In another embodiment, a nucleic acid encoding aneuregulin protein, or a functional fragment thereof, is administered.Any suitable route of administration can be used. Preferably,intravenous administration is used.

The present methods can further comprise administering a prophylactic ortherapeutic agent for myocardial infarction. Any suitable prophylacticor therapeutic agent can be used in the present methods. For example,the prophylactic or therapeutic agent used in the present methods can bean angiotensin I-converting enzyme inhibitor (ACEI), a calciumantagonist, a β-receptor antagonist, aspirin, atropine, nitroglycerin,scopolamine or a thrombolytic agent.

Any suitable ACEI can be used. Exemplary ACEIs include Captopril,Rampril, Lisinopril, Zofenopril and Trandolapril. Any suitable calciumantagonist can be used. Exemplary calcium antagonists include diltiazem.Any suitable β-receptor antagonist can be used. Exemplary β-receptorantagonists include propranolol, metoprolol, atenolol and timolol. Anysuitable thrombolytic agent can be used. Exemplary thrombolytic agentsinclude streptokinase, t-PA and anistreplase.

In a preferred embodiment, the neuregulin protein, or a functionalfragment thereof, the nucleic acid encoding a neuregulin protein, or afunctional fragment thereof, or the agent that enhances productionand/or function of the neuregulin is administered in vivo.

F. PHARMACEUTICAL COMPOSITIONS WITH PREFERRED SAFETY DOSAGE AND/ORREGIMEN

In one aspect, the present invention is directed to a pharmaceuticalcomposition for preventing, treating or delaying a disease in a mammal,which composition comprises a neuregulin protein, or a functionalfragment thereof: a) in a safety dosage equals to or less than about 170U/kg; or b) in a total regimen equals to or less than about 3,600 U/kg.

The present pharmaceutical compositions can be used in any suitableregimen and/or administration plans. In one example, the presentpharmaceutical compositions are administered for about 21 days or lessthan 21 days. In another example, the present pharmaceuticalcompositions are administered continuously or intermittently.

The present pharmaceutical compositions can be administered alone.Preferably, the present pharmaceutical compositions can further comprisea pharmaceutically acceptable carrier or excipient.

Any suitable neuregulin can be used in the present pharmaceuticalcompositions. For example, the neuregulin used in the presentpharmaceutical compositions can carry out its anti-disease activity viabinding with ErbB2-ErbB4 receptors. In another example, the neuregulinused in the present pharmaceutical compositions is neuregulin 1,neuregulin 2, neuregulin 3 or neuregulin 4. Preferably, the neuregulin 1is neuregulin α2 or neuregulin β2. In still another example, theneuregulin used in the present pharmaceutical compositions is aneuregulin β2 fragment comprising an amino acid sequence set forth inSEQ ID NO:4. Any specific neuregulin described in the above sections canalso be used.

The present pharmaceutical compositions can be used to prevent, treat ordelay a disease in any suitable mammal. Exemplary mammals include mice,rats, rabbits, cats, dogs, pigs, cows, ox, sheep, goats, horses, monkeysand other non-human primates. Preferably, the present methods are usedto prevent, treat or delay a disease in a human.

The present pharmaceutical compositions can be used to prevent, treat ordelay any suitable disease. Preferably, the present pharmaceuticalcompositions are used to, prevent, treat or delay a cardiovasculardisease, e.g., viral myocarditis, DCM, cardiotoxic activity, myocardialinfarction activity, etc.

In a preferred embodiment, the present pharmaceutical compositions areformulated for intravenous administration.

The following is an exemplary, rapid, sensitive, high flux andquantitative method for determination of biological activity of NRG-1through combining Neuregulin with cell surface ErbB3/ErbB4 molecule andindirect mediation of ErbB2 protein phosphorylation (See e.g., MichaelD. Sadick et al. Analytical Biochemistry, 1996, 235, 207-214). Accordingto the practical example 3 of the in vitro NRG-1 activity determinationas described in Michael D. Sadick et al., NGR-1 biological activity ofvarious origins can be determined.

Briefly, the assay, termed a kinase receptor activation enzyme-linkedimmunosorbant assay (KIRA-ELISA), consists of two separate microliterplates, one for cell culture, ligand stimulation, and celllysis/receptor solubilization and the other plate for receptor captureand phosphotyrosine ELISA. The assay was developed for analysis ofheregulin-induced ErbB2 activation and utilizes the stimulation ofintact receptor on the adherent breast carcinoma cell line, MCF-7.Membrane proteins are solubilized via Triton X-100 lysis and thereceptor is captured in ELISA wells coated with ErbB2-specificantibodies with no cross-reaction to ErbB3 or ErbB4. The degree ofreceptor phosphorylation is then quantified by antiphosphotyrosineELISA. A reproducible standard curve is generated with a EC (50) ofapproximately 360 pM for heregulin beta 1(177-244) (HRG beta 1(177-244).When identical samples of HRG beta 1(177-244) are analyzed by both theKIRA-ELISA and quantitative antiphosphotyrosine Western blot analysis,the results correlate very closely with one another. The assay describedin this report is able to specifically quantify tyrosine phosphorylationof ErbB2 that results from the interaction of HRG with ErbB3 and/orErbB4.

Since most of the genetically engineered medicines are proteins andpolypeptides, their activity can be determined by their amino acidsequences or the activity center formed by their spatial structure.Activity titer of protein and polypeptide is not consistent with theirabsolute quality, therefore cannot be determined with weight unit asthat of chemical drugs. However, biological activity of geneticallyengineered medicines is generally consistent with their pharmacodynamicsand titer determination system established through given biologicalactivity can determine its titer unit. Therefore, biological activitydetermination can be part of a process of titcring the substance withbiological activity and is an important component of quality control ofgenetically engineered product. It is important to determine biologicalactivity criteria for quality control of genetically engineered productand clinically used drugs.

Quantity of standard product that can induce 50% maximal reaction isdefined as an activity unit (1U). Accordingly, product from differentpharmaceuticals and of different batch number can be quantitated withuniform criteria.

G. THE FORMULATION, DOSAGE AND ROUTE OF ADMINISTRATION OF NEUREGULIN

The formulation, dosage and route of administration of a neuregulinprotein, or a functional fragment thereof, or a nucleic acid encoding aneuregulin protein, or a functional fragment thereof, preferably in theform of pharmaceutical compositions, can be determined according to themethods known in the art (see e.g., Remington: The Science and Practiceof Pharmacy, Alfonso R. Gennaro (Editor) Mack Publishing Company, April1997; Therapeutic Peptides and Proteins: Formulation, Processing, andDelivery Systems, Banga, 1999; and Pharmaceutical FormulationDevelopment of Peptides and Proteins, Hovgaard and Frkjr (Ed.), Taylor &Francis, Inc., 2000; Medical Applications of Liposomes, Lasic andPapahadjopoulos (Ed.), Elsevier Science, 1998; Textbook of Gene Therapy,Jain, Hogrefe & Huber Publishers, 1998; Adenoviruses: Basic Biology toGene Therapy, Vol. 15, Seth, Landes Bioscience, 1999; BiopharmaceuticalDrug Design and Development, Wu-Pong and Rojanasakul (Ed.), HumanaPress, 1999; Therapeutic Angiogenesis: From Basic Science to the Clinic,Vol. 28, Dole et al. (Ed.), Springer-Verlag New York, 1999). Theneuregulin protein, or a functional fragment thereof, or a nucleic acidencoding the neuregulin protein, or a functional fragment thereof, canbe formulated for oral, rectal, topical, inhalational, buccal (e.g.,sublingual), parenteral (e.g., subcutaneous, intramuscular, intradermal,or intravenous), transdermal administration or any other suitable routeof administration. The most suitable route in any given case will dependon the nature and severity of the condition being treated and on thenature of the particular neuregulin protein, or a functional fragmentthereof, or a nucleic acid encoding the neuregulin protein, or afunctional fragment thereof, which is being used.

The neuregulin protein, or a functional fragment thereof, or a nucleicacid encoding the neuregulin protein, or a functional fragment thereof,can be administered alone. Alternatively and preferably, the neuregulinprotein, or a functional fragment thereof, or a nucleic acid encodingthe neuregulin protein, or a functional fragment thereof, isco-administered with a pharmaceutically acceptable carrier or excipient.Any suitable pharmaceutically acceptable carrier or excipient can beused in the present method (See e.g., Remington: The Science andPractice of Pharmacy, Alfonso R. Gennaro (Editor) Mack PublishingCompany, April 1997).

The nucleic acid encoding a neuregulin protein, or a functional fragmentthereof, can be used in the form of naked DNA, complexed DNA, cDNA,plasmid DNA, RNA or other mixtures thereof as components of the genedelivery system. In another embodiment, the nucleic acid encoding aneuregulin protein, or a functional fragment thereof, is included in aviral vector. Any viral vectors that are suitable for gene therapy canbe used. For example, an adenovirus vector (U.S. Pat. No. 5,869,305), asimian virus vector (U.S. Pat. No. 5,962,274), a conditionallyreplicating human immunodeficiency viral vector (U.S. Pat. No.5,888,767), retrovirus, SV40, Herpes simplex viral amplicon vectors andVaccinia virus vectors can be used. In addition, the genes can bedelivered in a non-viral vector system such as a liposome wherein thelipid protects the DNA or other biomaterials from oxidation during thecoagulation.

According to the present invention, the neuregulin protein, or afunctional fragment thereof, or a nucleic acid encoding the neuregulinprotein, or a functional fragment thereof, alone or in combination withother agents, carriers or excipients, may be formulated for any suitableadministration route, such as intracavernous injection, subcutaneousinjection, intravenous injection, intramuscular injection, intradermalinjection, oral or topical administration. The method may employformulations for injectable administration in unit dosage form, inampoules or in multidose containers, with an added preservative. Theformulations may take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and may contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient may be in powder form for constitution with a suitablevehicle, sterile pyrogen-free water or other solvents, before use.Topical administration in the present invention may employ the use of afoam, gel, cream, ointment, transdermal patch, or paste.

Pharmaceutically acceptable compositions and methods for theiradministration that may be employed for use in this invention include,but are not limited to those described in U.S. Pat. Nos. 5,736,154;6,197,801 B1; 5,741,511; 5,886,039; 5,941,868; 6,258,374 B1; and5,686,102.

The magnitude of a therapeutic dose in the treatment or prevention willvary with the severity of the condition to be treated and the route ofadministration. The dose, and perhaps dose frequency, will also varyaccording to age, body weight, condition and response of the individualpatient.

It should be noted that the attending physician would know how to andwhen to terminate, interrupt or adjust therapy to lower dosage due totoxicity, or adverse effects. Conversely, the physician would also knowhow to and when to adjust treatment to higher levels if the clinicalresponse is not adequate (precluding toxic side effects).

Any suitable route of administration may be used. Dosage forms includetablets, troches, cachet, dispersions, suspensions, solutions, capsules,patches, and the like. See, Remington's Pharmaceutical Sciences.

In practical use, the neuregulin protein, or a functional fragmentthereof, or a nucleic acid encoding the neuregulin protein, or afunctional fragment thereof, alone or in combination with other agents,may be combined as the active in intimate admixture with apharmaceutical carrier or excipient, such as beta-cyclodextrin and2-hydroxy-propyl-beta-cyclodextrin, according to conventionalpharmaceutical compounding techniques. The carrier may take a wide formof preparation desired for administration, topical or parenteral. Inpreparing compositions for parenteral dosage form, such as intravenousinjection or infusion, similar pharmaceutical media may be employed,water, glycols, oils, buffers, sugar, preservatives, liposomes, and thelike known to those of skill in the art. Examples of such parenteralcompositions include, but are not limited to dextrose 5% w/v, normalsaline or other solutions. The total dose of the neuregulin protein, ora functional fragment thereof, or a nucleic acid encoding the neuregulinprotein, or a functional fragment thereof, alone or in combination withother agents to be administered may be administered in a vial ofintravenous fluid, ranging from about 1 ml to 2000 ml. The volume ofdilution fluid will vary according to the total dose administered.

The invention also provides for kits for carrying out the therapeuticregimens of the invention. Such kits comprise in one or more containerstherapeutically effective amounts of the neuregulin protein, or afunctional fragment thereof, or a nucleic acid encoding the neuregulinprotein, or a functional fragment thereof, alone or in combination withother agents, in pharmaceutically acceptable form. Preferredpharmaceutical forms would be in combination with sterile saline,dextrose solution, or buffered solution, or other pharmaceuticallyacceptable sterile fluid. Alternatively, the composition may belyophilized or dessicated; in this instance, the kit optionally furthercomprises in a container a pharmaceutically acceptable solution,preferably sterile, to reconstitute the complex to form a solution forinjection purposes. Exemplary pharmaceutically acceptable solutions aresaline and dextrose solution.

In another embodiment, a kit of the invention further comprises a needleor syringe, preferably packaged in sterile form, for injecting thecomposition, and/or a packaged alcohol pad. Instructions are optionallyincluded for administration of composition by a physician or by thepatient.

H. EXAMPLES

Recombinant Human Neuregulin-1 β_(S177-Q237) (rhNRG-1 β_(S177-Q237)Neuregulin-1) developed by the present inventor can repair damagedmyocardial cell structure, strengthen connection between these cells,improve myocardial function and strengthen myocardial biological effect.The experiments described herein demonstrate that certain Neuregulinfragments, e.g., rhNRG-1 β_(S177-Q237), can effectively treat variousforms of cardiovascular disease such as viral myocarditis or dilated(congestive) cardiomyopathy (DCM), cardiotoxic caused by certaintherapeutic agents, or myocardial infarction in vivo and do not affecthemodynamics of normal animals

Example 1 Recombinant Production of rhNRG-1 β_(S177-Q237) FIG. 1.Technical Outline of Construction of an Engineered Strain

Human neuregulin gene is located in chromosome 8P12 with about 13 exons.Recombinant neuregulin fragment is composed of 61 amino acid.Theoretical molecular weight is 7,055 D. Apparent molecular weight inSDS-PAGE electrophoresis is 6,500-7,000 D. Its isoelectric point isabout 6.5. There is no glycosylated locus. The peptide structurecontains 3 disulfide bonds. This gene is suitable for expression in E.coli.

PET22b was selected as expression plasmid. Human neuregulin gene wasintroduced into the plasmid and then E. coli BL21 was transformed bythis plasmid. High level expression recombinant was screened out asengineered strain for producing recombinant human neuregulin fragment.

FIG. 2. Amplification of Human Neuregulin Gene

Total RNA and mRNA were extracted from brain tissue of 5-month humanfetus and reversibly transcribed to cDNA. RT-PCR was performed with thetranscribed cDNA as template, and a pair of primers P1, TCG AAC ATA TGAGCC ATC TTG TAA AAT GTG CGG (SEQ ID NO:1) and P2, TCG AAG GGC CCT CACTGG TAC AGC TCC TCC (SEQ ID NO:2) to amplify target gene. The PCRproduct was examined in electrophoresis on 1.5% agarose. Specific 183 bpDNA fragment was found in agarose, the length of which was the same asanticipated.

FIG. 3. Physical Map of Plasmid PET22b FIG. 4. Identification ofRecombinant Plasmid by Endonuclease Digestion

Calcium chloride sedimentation method was applied to clone humanneuregulin gene into the expression plasmid PET22b to constructrecombinant human neuregulin expression plasmid (PET22b-humanneuregulin). This gene was expressed efficiently under the drive of T7promoter. N-terminal of expressed gene was inserted at NdeI locus.C-terminal terminator is next to the last amino acid. The expressedprotein did not form fusion protein with any amino acid. An accurate 183bp fragment was obtained after endonuclease digestion analysis.Transformant was characterized by endonuclease digestion.Double-stranded DNA was extracted for sequence analysis. The sequencingresults confirmed that the sequence of human neuregulin carried inexpression vector was completely correct. The determined cDNA sequenceis listed here: AGC CAT CTT GTA AAA TGT GCG GAG AAG GAG AAA ACT TTC TGTGTG AAT GGA GGG GAG TGC TTC ATG GTG AAA GAC CTT TCA AAC CCC TCG AGA TACTTG TGC AAG TGC CCA AAT GAG TTT ACT GGT GAT CGC TGC CAA AAC TAC GTA ATGGCC AGC TTC TAC AAG GCG GAG GAG CTG TAC CAG (SEQ ID NO:3). The deducedamino acid sequence based on the above cDNA sequence is:SHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASF YKAEELYQ (SEQ IDNO:4).

FIG. 5. Screening for Expression of an Engineered Strain

After PCR amplification and endonuclease digestion analysis, singlecolony of the engineering clone (BL21-PET22b-human-neuregulin) wasrandomly picked to inoculate in 2 ml LB-Amp liquid medium. The culturewas incubated overnight at 37° C. and shaking at 250 rpm. Then aproportion of pure culture was inoculated into 20 ml LB-Amp medium. Theculture was collected after incubation at 37° C. till the turbidityincreased to 1.0 at OD₆₀₀ and after IPTG was added for 4 h to induce theexpression. Inclusion body was collected after the cells weredestructed. After electrophoresis in 15% SDS-PAGE, thin-layer scanninganalysis, Western-blotting, and repeated screening, an engineered strain(BL21-PET22b-human neuregulin) was characterized and established withstable high level expression of target protein neuregulin. The expressedtarget protein accounted for approximately 10% of the total protein inthis strain. After high-pressure homogenate process, the target proteinpresented in the form of inclusion body.

The engineered strain was analyzed by SDS-PAGE electrophoresis after 4 hinduction by IPTG. Inclusion body accounted for about 20% of the totalproteins. Purified recombinant neuregulin specific activity was morethan 5×10³ EU/mg, indicating that the construction of neuregulinproducing strain was successful. In addition to the SDS-PAGE, WesternBlot, biological activity analysis at the stage of strain screening,further analysis was carried out, such as neuregulin amino acidcomposition analysis and N-terminal sequence analysis. These resultsindicate that the amino acid sequence of expressed recombinant humanneuregulin is the same as designed.

Example 2 Therapeutic Effect of Recombinant Human Neuregulin-1 β onHeart Failure in Rat Caused by Ligation of Coronary Artery 1. Abstract

Objectives To study the therapeutic effect of Recombinant HumanNeuregulin-1 β (rhNRG-1β) on heart failure in rat caused by ligation ofcoronary artery. Method Open the chest of rat, descending limb of thecoronary was ligated with non-invasive sutures at the site between leftauricle and pulmonary cone, set up a subacute heart failure rat model.In general, about 6 days after the ligation when ejection fraction ofthe left ventricle decreased by about 50%,

The groups were randomly divided, i.e., model group, testing drug groupand pseudo-operation group that only opened the chest but did not ligatethe coronary artery. There were 10-13 animals in each group, 3 dosagelevel groups were set up for the testing drug group, they were 5, 10 and20 μg/kg respectively, the drug was injected into the tail vein onceevery day for consecutive 10 days. Heart function determination(echocardiography) was performed 6 days prior to the drug administrationand after the drug withdrawal, the testing animals autopsied, heartweight measured, ventricular wall thickness determined, pathologicexamination and plasma renin-angiotensin-aldosterone level determinedResults After consecutive 5-day of drug administration in 3 dosage levelgroups of rhNRG-1 β, ejection fraction (71.1±12.0%, 64.4±12.9%,62.9±8.4%) and shortening fraction (36.9±9.7%, 32.0±9.5%, 30.3±6.1%) ofthe model animals were all increased respectively and, there wassignificant difference between that of the 20 μg/kg, 10 μg/kg groupcompared with the model group (P<0.01); in addition, the changes ofejection fraction in the 20 μg/kg model group could maintain for about35 days after the drug administration (P<0.05); 20 μg/kg of rhNRG-1 βcould significantly reduce ischemic area of the myocardium, increasecapillary number of the fibrotic lesion (P<0.05); 20 μg/kg and 10 μg/kgof rhNRG-1 β could reduce peripheral angiotensin I (AI), angiotensin II(AII), renin (PRA) and aldosterone (ALD) levels and with significantdifference when comparing with that of the model group (P<0.01, P<0.05);There wasn't significant difference between group with consecutive 10days of injection and group with consecutive 5 days of injection(P<0.05). Conclusion Certain dosage of rhNRG-1 β injected intravenouslyfor consecutive 5 days could effectively treat heart failure in ratcaused by ligation of coronary artery.

2. Objectives of the Experiment

To study the therapeutic effect of rhNRG-1 β on heart failure in ratcaused by ligation of coronary artery.

3. Testing Drug

rhNRG-1 β researched and produced by Zensun (Shanghai) Science &Technology Co Ltd, batch number: 200110006-2; concentration: 500μg/ampule; titer determination: 5000 u/amplule; purity: >95% (HPLC-C8).

4. Experiment Animal

4.1 Species, sources and number of certificate of competency: SD rat,provided by Experimental Animal Center of Chinese Academy of Science,number of certificate of competency: Zhong Ke Yuan Dong Guan Hui 003;4.2 Body weight, gender: 200-220 g, male.

5. Reagents and Equipments

-   5.1 Echocardiograph device, Hewlett Packard Sonos 5500, type of    probe:S12;-   5.2 Six leads electro-physiology recorder, SMUP-C-6, manufactured by    Physiology Department of Shanghai Medical University;-   5.3 Electro-balance, Mateler-Tolido Equipment (Shanghai) Co Ltd;-   5.4 Arterial-venous indwelling needle, 20 G, produced by    Sino-America Weng Zhou Hua Li Medical Equipment Company;-   5.5 Micro-vernier calipers, Harbin Measure & Knife Factory;-   5.6 Radio-immune γ counter, GC-1200, Zhong Jia Photoelectric    Equipment Branch of General Science & Technology Industry Company of    China Science & Technology University;-   5.7 Renin, angiotensin kits, AI: Beijing North Bio-Tech Institute,    batch number: 0210; AII: Beijing North Bio-Tech Institute, batch    number: 2028;-   5.8 Depilatory, 8% sodium sulfide, Xi Long Chemical Plant of    GuangDong Province, batch number: 010622;-   5.9 Ketamine hydrocloride, manufactured by Shanghai Zhong Xi    Pharmaceuticals Co Ltd, batch number: 20020401.

6. Method of the Experiment 6.1 Experiment Grouping

Pseudo-operation group, model group and testing drug groups were set up.

Pseudo-operation group (n=10), i.e. thoracotomy but without ligating thecoronary artery;

Model group (negative control group) (n=12): vehicle of the preparationwas injected (10 mM PB, 0.2% human serum albumin, 5% mannitol);

Testing drug group (n=13): rhNRG-1 β with 3 dosage level, 2 subgroupswere established for each dosage level group; in which, one group servedas long-term monitoring after the drug administration.

6.2 Dosage Set Up of the Testing Drug and Drug Preparation, Route ofDrug Administration, Times of Injection, Concentration and Volume of theAdministered Drug

High, medium and low dosage level group of 20 μg/kg, 10 μg/kg and 5μg/kg respectively were set up according to the result of preliminaryexperiment. The drug was diluted with preparation buffer solution (10 mMPB, 0.2% human serum albumin, 5% mannitol) to the needed concentration.

Route of drug administration: in both testing drug group and model groupthe drug was injected into the tail vein once per day for consecutive 5days. Echocardiography was performed at the 6^(th) day prior to drugadministration, then continuously injected for 5 days. Volume of eachdrug injection was 0.4 ml/100 g body weight.

6.3 Method of the Experiment 6.3.1 Set Up Heart Failure Rat ModelThrough Ligation of Anterior Descending Limb of the Coronary Artery

After anesthesia with intra-abdominal injection of 100 mg/kg ketamine,the rat was fixed on rat plate in supine position, disinfection of theneck region with bromo-geramine was carried out after depilating.Midline incision was made, trachea was found after separating theanterior cervical muscle, 18 G arterial indwelling trochar was insertedinto the trachea at the level of 3-5 tracheal cartilage, took out thestylet, push the plastic cannula 1-2 cm further into the trachea, fixedit for later connection with ventilator for small animal (tidal volumewas about 20 ml, frequency was 80/min). After cut open the left anteriorchest wall skin, separated the muscle, exposed the 4^(th) and 5^(th)rib, penetrate the chest wall with curve forceps, separated tissue underthe ribs, connected with ventilator, exposed the heart, monitoring theinflation of the lungs and the heart beat, tore open the pericardium,turned the upper fat pad up, full exposed the left auricle and pulmonarycone, ligated the left coronary artery between the two parts with 6/0non-invasive suture for medical use, infarcted myocardial area (about 8mm×8 mm) showed violet color, protruding and with significantly reducedactivity after the ligation. Then closed the chest wall, block theopening of the ventilator to inflate the lungs, forcefully pressed thechest to drive out the air, then sutured the chest muscle and skin.Monitoring the respiration, after spontaneous respiration recovered,withdrawn the ventilator, took the animal back to its cage for breeding.In the pseudo-operation group, only the pericardium was tore open, butwithout the coronary artery ligation.

Echocardiograph was performed for the operated rat at about 6 dayspostoperatively, those animals with about 50% reduction of EF value wererandomly divided into groups, the EF value of each group was all about50%, then drug administration began.

6.3.2 Pharmacodynamic Experiment

Those animals with EF value of about 50% were randomly divided intomodel group (negative group) and 3-dosage level testing drug groups with12-13 animals in each group. Route of drug administration in testingdrug group and model group was injection into the tail vein, once everyday for consecutive 5 days. After echocardiography at the 6^(th) day,another 5 days of consecutive drug administration was carried out. Drugvolume of each injection was 0.4 ml/100 g body weight.

Echocardiography was again performed after the end of drugadministration, the animals autopsied, heart weight measured, thicknessof ventricular wall determined, pathologic examination of the heartperformed, peripheral blood collected, plasma separated andrenin-angiotensin-aldosterone determined.

Some testing animals of various groups were left out for long-termmonitoring.

6.3.3 Observation Index 6.3.3.1 Heart Function Determination

Echocardiography was performed under anesthesia with ketamine after thelegation and prior to drug administration and at the 6^(th) day, 11^(th)day of drug administration, the major index included:

-   -   EF: heart ejection fraction, reflecting ejection function of        ventricle;    -   FS: ventricular short axis shortening rate, reflecting        contraction function of ventricle;    -   LVDd: diastolic maximal inner diameter of left ventricle (cm);    -   LVDs: systolic minimal inner diameter of left ventricle (cm).

6.3.3.2 Determination of the Content of PlasmaRenin-Angiotensin-Aldosterone

It was submitted to be performed by Radio-isotope Department of ZhongShan Hospital affiliated to Fudan University.

Carotid artery phlebotomy was carried out and plasma extracted accordingto the mandate of the reagent kit, frozen and stored at −20° C. Reninactivity (PRA), angiotensin I (AI), angiotensin II (AII) and aldosterone(ALD) content were determined with immunoassay.

6.3.3.3 Pathohistological Section of the Myocardium

It was performed by Professor Wand Bing Seng of Shanghai DifficultPathologic Consulting Center.

After fixed with 10% formaldehyde, myocardium of the rat waslongitudinally cut with equal distance into 3 slices, conventionallyembedded with paraffin, section made, HE stained, image analysis forarea of fibrotic region of the heart, counted the number of capillary inthe fibrotic lesion (counting unit: piece/mm²); Nagar-Oslen stained,studied the size of ischemic hypoxic region in the myocardium.

7. Data Processing

t test was carried out for the experimental data collected.

8. Result of the Experiment

8.1 Effect of rhNRG-1 β on Function of the Ischemic Heart in Rat

Results of echocardiography performed at 5-day of drug administrationshowed that EF value (50.2±8.4%) and FS value (22.4±4.6%) of the modelgroup was significantly lower than EF (91.1±2.4%) and FS (57.3±3.9%) ofthe pseudo-operation group and with significant difference (P<0.01).Both EF value (71.1±12.0%, 64.3±12.8%, 62.9±8.4%) and FS value(36.9±9.7%, 32.0±9.5%, 30.3±6.1%) of testing drug groups (20, 10, and 5μg/kg) increased significantly once again, and there was significantdifference when comparing with those of the model group (P<0.01).

Results of repeated echocardiography performed at 10-day of drugadministration showed that EF value (42.7±6.4%) and FS value (18.3±3.2%)were still significantly lower than EF (95.0±2.8%) and FS (65.3±6.8%) ofthe pseudo-operation group and with significant difference (P<0.01).While EF value (75.7±10.8%, 61.4±15.0%, 59.2±12.4%) and FS value(41.3±11.0%, 30.3±10.4%, 28.4±8.6%) of the testing drug groups (20, 10,5 μg/kg) still maintained at relative high level, and there wassignificant difference when comparing with those of the model group(P<0.01), however, there was no significant difference when comparingwith the results of 5-day of drug administration. Tables 1-6 showed theresults of the two experiments.

Observation on EF value of rat heart in the model animal group andtesting drug group at 5-day of drug administration and 35-day after drugwithdrawal were carried out, the result showed that rat heart EF valueof the 20 μg/kg rhNRG-1 β group maintained stable at relatively highlevel and there was significant difference when comparing with that ofthe model animal group (P<0.05) (Table 7).

TABLE 1 Measurement parameters of heart function prior to drugadministration in ligation of anterior descending limb of the coronaryartery produced heart failure model animals (1) Group LVDd LVDs EF FSPseudo-operation group 0.572 ± 0.258 ± 89.5 ± 55.2 ± (n = 6) 0.033*0.046** 4.4** 5.8** Model group(n = 12) 0.705 ± 0.558 ± 48.3 ± 21.3 ±0.117 0.119 10.3 5.6 rhNRG-1 β group 0.730 ± 0.575 ± 49.3 ± 21.7 ± 20μg/kg (n = 13) 0.108 0.119 11.4 6.4 rhNRG-1 β group 0.709 ± 0.555 ± 49.5± 22.1 ± 10 μg/kg (n = 13) 0.099 0.102 11.0 6.1 rhNRG-1 β group 0.761 ±0.596 ± 49.3 ± 22.0 ± 5 μg/kg (n = 13) 0.075 0.092 10.6 5.9 *P < 0.05,**<0.01, when comparing with that of the model animal group

TABLE 2 Measurement parameters of heart function 5 days after the drugadministration in ligation of anterior descending limb of the coronaryartery produced heart failure model animals (1) Drug adminis- Grouptration LVDd LVDs EF FS Pseudo-operation iv 0.549 ± 0.234 ± 91.1 ± 57.3± group(n = 6) qd × 5 0.046** 0.027** 2.4** 3.9** Model group iv 0.79 ±0.61 ± 50.24 ± 22.43 ± (n = 12) qd × 5 0.08 0.09 8.41 4.62 rhNRG-1 β iv0.70 ± 0.46 ± 71.07 ± 36.88 ± 20 μg/kg (n = 13) qd × 5 0.07** 0.09**11.99** 9.66** rhNRG-1 β iv 0.71 ± 0.51 ± 64.35 ± 32.01 ± 10 μg/kg (n =13) qd × 5 0.05* 0.09** 12.85** 9.54** rhNRG-1 β iv 0.73 ± 0.54 ± 62.90± 30.32 ± 5 μg/kg (n = 11) qd × 5 0.05* 0.09* 8.39** 6.11** *p < 0.05,**p < 0.01, when comparing with that of the model group

TABLE 3 Measurement parameters of heart function 10 days after the drugadministration in ligation of anterior descending limb of the coronaryartery produced heart failure model animals (1) Drug adminis- Grouptration LVDd LVDs EF FS Pseudo-operation iv 0.466 ± 0.159 ± 95.0 ± 65.3± (n = 6) qd × 10 0.041** 0.036** 2.8** 6.8** Model group 1 iv 0.8 ± 0.7± 42.7 ± 18.3 ± (n = 10) qd × 10 0.11 0.11 6.36 3.19 rhNRG-1 β iv 0.6 ±0.5 ± 75.7 ± 41.3 ± 20 μg/kg (n = 10) qd × 10 0.12** 0.14** 10.78**10.98** rhNRG-1 β iv 0.7 ± 0.51 ± 61.4 ± 30.3 ± 10 μg/kg (n = 10) qd ×10 0.07** 0.14** 15** 10.36** rhNRG-1 β iv 0.72 ± 0.55 ± 59.2 ± 28.4 ± 5μg/kg (n = 10) qd × 10 0.10* 0.12* 12.37** 8.62** *p < 0.05, **p < 0.01,when comparing with that of the model group

TABLE 4 Measurement parameters of heart function 10 days after the drugadministration in ligation of anterior descending limb of the coronaryartery produced heart failure model animals (II) Group LVDd LVDs EF FSPseudo-operation group 0.544 ± 0.215 ± 93 ± 60.8 ± (n = 6) 0.071*0.053** 2.9** 5.2** Model group (n = 12) 0.66 ± 0.52 ± 46.98 ± 20.85 ±0.10 0.11 14.17 7.38 rhNRG-1 β 20 μg/kg 0.74 ± 0.56 ± 52.29 ± 23.89 ± (n= 12) 0.12 0.11 12.87 7.28 rhNRG-1 β 10 μg/kg 0.64 ± 0.49 ± 51.67 ±23.27 ± (n = 12) 0.11 0.11 11.92 6.73 rhNRG-1 β 5 μg/kg 0.66 ± 0.51 ±50.81 ± 22.83 ± (n = 12) 0.11 0.12 12.55 6.92 *p < 0.05, **p < 0.01,when comparing with that of the model group

TABLE 5 Measurement parameters of heart function 5 days after the drugadministration in ligation of anterior descending limb of the coronaryartery produced heart failure model animals (II) Drug adminis- Grouptration LVDd LVDs EF FS Pseudo-operation iv 0.553 ± 0.215 ± 93.1 ± 61.4± group (n = 6) qd × 5 0.063** 0.052** 3.9** 6.2** Model group iv 0.87 ±0.74 ± 38.00 ± 16.35 ± (n = 10) qd × 5 0.11 0.14 12.36 5.55 rhNRG-1 βi.v 0.68 ± 0.46 ± 65.47 ± 34.23 ± 20 μg/kg qd × 5 0.11** 0.17** 20.48**15.42** (n = 9) rhNRG-1 β iv 0.72 ± 0.54 ± 56.51 ± 26.53 ± 10 μg/kg qd ×5 0.13** 0.14** 12.68** 8.48** (n = 12) rhNRG-1 β iv 0.74 ± 0.58 ± 56.76± 28.35 ± 5 μg/kg qd × 5 0.11* 0.18* 16.10** 10.64** (n = 10) *p < 0.05,**p < 0.01, when comparing with that of the model group

TABLE 6 Measurement parameters of heart function 10 days after the drugadministration in ligation of anterior descending limb of the coronaryartery produced heart failure model animals (II) Drug adminis- Grouptration LVDd LVDs EF FS Psudo-operation iv 0.539 ± 0.204 ± 93.8 ± 62.2 ±group (n = 6) 10qd 0.015** 0.017** 1.6** 3.6** Model group iv 0.81 ±0.69 ± 36.13 ± 15.18 ± (n = 10) 10qd 0.13 0.13 10.10 5.01 rhNRG-1 β iv0.65 ± 0.42 ± 70.22 ± 36.49 ± 20 μg/kg 10qd 0.09** 0.13** 14.15**11.27** (n = 8) rhNRG-1 β iv 0.68 ± 0.49 ± 66.54 ± 34.28 ± 10 μg/kg 10qd0.08** 0.17** 15.81** 12.64** (n = 12) rhNRG-1 β iv 0.70 ± 0.53 ± 57.90± 29.26 ± 5 μg/kg 10qd 0.08* 0.16* 19.54** 14.19* (n = 9) *p < 0.05, **p< 0.01, when comparing with that of the model group

TABLE 7 EF value of heart function measured at 5-day of drugadministration and 35-day after drug withdrawal in model animals Afterligation 5-day after drug prior to drug 5-day after drug administrationand 35-day administration administration after drug withdrawal rhNRG-1 β20 μg/kg 52.4 ± 13.8 79.2 ± 4.7*  73.0 ± 20.3* rhNRG-1 β 10 μg/kg 52.2 ±12.8 77.3 ± 11.6 63.8 ± 23.8 rhNRG-1 β 5 μg/kg 53.1 ± 12.8 75.8 ± 15.369.9 ± 28.6 Model group 53.0 ± 12.6 62.6 ± 16.7 52.5 ± 29.9Pseudo-operation  94.4 ± 3.7**  94.1 ± 2.5**  95.1 ± 2.3** group *p <0.05; **p < 0.01, when comparing with that of the model animal group8.2 Effect of rhNRG-1 β on Pathohistology of Model Rat Myocardium8.2.1 Effect of rhNRG-1 β on the Severity of Myocardial Ischemia andHypoxia in Model Animals

In Nagar-Oslen stained section, normal myocardium stained with lightyellow color, while ischemic hypoxic area stained deeper with reddishcolor. Ischemic hypoxic changes could be analyzed with this method andcomparison of qualitative and semi-quantitative test could be made.

FIGS. 6-15 showed the pathohistological changes of the various testinggroup. Myocardium of the model group stained deep color, large patch ofmyocardium was stained with red color, while that of thepseudo-operation group stained yellow, without red stained area; redstained myocardial cells of 20 μg/kg rhNRG-1 β showed punctuatedistribution; red stained tissue of 10 μg/kg and 5 μg/kg groups showedpatchy distribution, demonstrating that rhNRG-1 β could alleviate theseverity of ischemic hypoxic damages.

8.2.2 Effect of rhNRG-1 β on Capillary Regeneration in the Fibrotic Areaof Ischemic Myocardium of Model Rat

Changes of capillary number in myocardial tissue of various testinggroups were studied with Leica image analysis system. The resultsrevealed that there was increase of capillary number in the fibroticlesion of myocardium of model animal in the testing drug group withrhNRG-1 β and there was significant difference between 20 μg/kg dosagelevel group and that of the model group (P<0.01), no statisticdifference was seen between 10 μg/kg and 5 μg/kg dosage level groups andthat of the model group, demonstrating that high dosage of rhNRG-1 β (20μg/kg) could promote capillary proliferation in myocardial fibroticlesion after ligation of the coronary artery in rat (Table 8, FIGS. 16and 17).

TABLE 8 Effect of rhNRG-1 β on capillary regeneration in myocardialfibrotic area of model animal Drug Capillary Capillary adminis- countingcounting Group tration (I) (II) Pseudo-operation group iv qd x 10 0 ± 0± 0 0 Model group iv qd x 10 6.49 ± 13.7 ± 2.17 5.5 rhNRG-1 β 20 μg/kgiv qd x 10 10.12 ± 24.6 ± 3.0** 9.5** rhNRG-1 β 10 μg/kg iv qd x 10 8.99± 19.6 ± 3.3 8.5 rhNRG-1 β 5 μg/kg iv qd x 10 9.35 ± 15.0 ± 4.2 6.9 **P< 0.01, when comparing with that of the model group8.3 Effect of rhNRG-1 β on Plasma Rennin-Angiotensin-Aldosterone Levelin Model Animal

Plasma rennin (PRA), angiotensin I (AT), angiotensin II (AII) andaldosterone (ALD) levels was determined in various testing drug groupswith radioimmunoassay. In the model animal group, the results showedthat PRA, AI, AII and ALD were 3.506±1.78 ng/ml·h, 10.655±1.18 ng/ml,1366.38±577.33 pg/ml and 1.738±0.34 ng/ml respectively; Comparing withthose of the pseudo-operation group (1.315±0.96 ng/ml, 8.125±1.57 ng/ml,564.37±273.56 pg/ml and 1.113±0.45 ng/ml), the content increasedsignificantly, the difference was significant statistically (P<0.05).

Plasma AI (7.40±12.15, 7.65±1.40 ng/ml), and AII (641.47±283.86,468.58±165.10 pg/ml) level reduced significantly in 20, and 10 μg/kgrhNRG-1 β group with significant difference (P<0.01); PRA (1.337±1.09,1.075±1.50 ng/ml·h) and ALD (1.02±0.27, 1.26±0.38 ng/ml) reducedsignificantly as well and there was significant difference whencomparing with that of the model animal group (P<0.05). However, whencomparing with that of the pseudo-operation group, the difference has nostatistical implication (P>0.05), demonstrating that certain dosagelevel of rhNRG-1 β could reduced plasma PRA, AI, AII and ALD content inrat after ligation of its coronary artery (Table 9).

TABLE 9 Plasma rennin (PRA), angiotensin I (AI), angiotensin II (A II)and aldosterone (ALD) in various testing group model animals Drugadminis- AI AII ALD PRA(ng/ Group tration (ng/ml) (Pg/ml) (ng/ml) ml ·h) Pseudo- iv 8.125 ± 564.370 ± 1.113 ± 1.315 ± operation qd × 10 1.573*273.56* 0.447* 0.96* n = 6 rhNRG-1 β iv 10.655 ± 1366.38 ± 1.738 ± 3.506± (n = 6) qd × 10 1.178 577.33 0.337 1.78 rhNRG-1 β iv 7.400 ± 641.47 ±1.018 ± 1.337 ± 20 μg/kg qd × 10 2.15** 283.86* 0.266** 1.09* (n = 6)rhNRG-1 β iv 7.654 ± 468.583 ± 1.264 ± 1.075 ± 10 μg/kg qd × 10 1.399**165.1** 0.382* 1.5* (n = 6) rhNRG-1 β iv 10.036 ± 807.304 ± 1.472 ±4.032 ± 5 μg/kg qd × 10 2.283 333.46 0.413 1.81 (n = 6) *p < 0.05, **p <0.01, when comparing with that of the model group

9. Conclusion

When comparing with the ejection fraction (50.2±8.4%) and shorteningfraction (22.4±4.6%) of the model control group, three dosage level ofrhNRG-1 β injected for consecutive 5 days could raise the ejectionfraction (71.1±12.0%, 64.4±12.9%, 62.9±8.4%) and shortening fraction(36.9±9.7%, 32.0±9.5%, 30.3±6.1%) and there was significant differencebetween 20 μg/kg, 10 μg/kg group and the model animal group (P<0.01), inaddition, changes of ejection fraction in testing drug group modelanimal could maintain for about 35 days after drug administration(P<0.05); 20 μg/kg of rhNRG-1 β could significantly reduce ischemichypoxic area of the myocardium, increase capillary number in thefibrotic lesion (P<0.05); 20 μg/kg and 10 μg/kg of rhNRG-1 β couldreduce angiotensin I (AI), angiotensin II (AII), rennin (PRA) andaldosterone (ALD) levels in the peripheral blood of model animals andwith significant difference when comparing with that of the model animalgroup (P<0.01, P<0.05). There was no significant difference betweenthose with consecutive 10 days of drug administration and those withconsecutive 5 days of drug administration (P<0.05).

Results of the experiment showed that certain dosage of 20 μg/kg ofrhNRG-1 β injected intravenously for consecutive 5 days couldeffectively treat rat heart failure caused by ligation of the coronaryartery.

Example 3 Therapeutic Effect of 20 μg/Kg of rhNRG-1 β on Heart FailureCaused by Adriamycin in SD Rat 1. Abstract

Objectives To study therapeutic effect of 20 μg/kg of rhNRG-1 β on toxicmyocarditis caused by Adriamycin in rat. Method 3.3 mg/kg of Adriamycinwas injected in SD rat's tail vein, once every week for consecutive 4injections, set up Adriamycin caused SD rat toxic myocarditis model.3-dosage levels of rhNRG-1 β groups were set up, they were 10, 20 and 40μg/kg once intravenous injection per day (qd) for consecutive 10 days.Survival of the animals was monitored; blood flow dynamic index, ratioof heart weight/body weight, pathologic examination of the myocardiumwere monitored at the end of the experiment, serum troponnin T (cTnT)level was determined as well. Results The survival rate of 40, 20 and 10μg/kg dosage level of rhNRG-1 β group raised significantly whencomparing with that of the 15% survival rate of the model animals,reached 85%, 90% and 60% respectively, dp/dt, −dp/dt and LVPmax of high,medium and low testing drug dosage level groups raised significantly,dp/dt reached 5954±689, 6107±418 and 4875±636 respectively, −dp/dt was−4794±954, −4323±457 and −3672±884 respectively, LVPmax was 165.7±22.7,156.1±17.7 and 145±15.2 respectively, there was significant differencewhen comparing with those of the model animal group (P<0.001), inaddition, there was significant difference in dp/dt, −dp/dt, LVPmaxbetween 40 and 20 μg/kg dosage groups and those of 10 μg/kg dosage groupP<0.05) and was somewhat dosage dependent; 40, 20 and 10 μg/kg ofrhNRG-1 β could effectively alleviate the severity of myocardial damagein model animals, reduced serum troponin T (eTnT) level, they were0.025±0.011, 0.031±0.006 and 0.074±0.024 respectively, with significantdifference when comparing with that of 0.205±0.072 of the model group(P<0.01). Conclusion 20 μg/kg of rhNRG-1 β could effectively treat toxicmyocardial damage caused by Adriamycin in rat.

2. Objectives of the Experiment

To study the therapeutic effect of rhNRG-1 β on toxic myocarditis causedby Adriamycin in rat.

3. Testing Drug

rhNRG-1 β, provided by Zensun (Shanghai) Science & TechnologyDevelopment Co. Ltd. Batch number: 200110006-2 Specification: 500μg/ampule. Titer determination: 5000 u/ampule; Purity:>95% (HPLC-C8).

4. Experiment Animal

SD rat: provided by Experimental Animal Center of Fudan UniversityMedical College, Number of certificate of animal competency: Yi Dong ZiFeb. 22, 2011. body weight 250±30 g, male.

The animals were randomly divided in groups, 20 animals in each group,bred in separate cage.

Temperature of the animal room was 18-22° C., with relative humidity of50%-70%.

5. Reagents and Equipment

Six leads electro-physiology recorder, SMUP-C-6, manufactured byPhysiology Department of Shanghai Medical University;Energy exchanger (Japan Photoelectricity Industry Company, NIHON KOHDEN,model: TP-400T);Electrochemistry luminescence automatic immune analysis device (model2010): Roche Diagnostics Co Ltd, batch number 158468;Precisive electro-balance, (Mateler-Tolido Equipment Co Ltd; Max: 610 gd=0.01 g);Micro-vernier calipers, (Harbin Measure & Knife Factory, 0.05 mm);Arterial-venous indwelling needle, 20 Gm produced by Sino-America WengZhou Hua Li Medical Equipment Company;Reagent kit for serum troponin determination (Behring Dignostic Inc.).

6. Method of the Experiment 6.1 Experiment Grouping

Normal control group, model animal group and testing drug group were setup;Model group (negative control group: Adriamycin, manufactured by MingZhiPharmaceuticals; ShanTou Special Economy Prefecture (batch number:000201, expired date: 2003February);Testing drug group: rhNRG-1β group.

6.2 Dosage Set Up, Preparation of Testing Drug, Route of DrugAdministration, Times of Drug Administration, Concentration and Volumeof the Testing Drug

3-dosage levels of 10, 20 and 40 μg/kg of the testing drug group wereestablished. Dissolved with 1 ml of water for injection and adjusted toneeded concentration with excipient. Major component of the excipientwas 5% mannitol for injection, 0.2% human serum albumin for injection,10 mM phosphate buffer solution, provided by Zensun (Shanghai) Science &Technology Development Co Ltd. The testing drug was prepared prior toinjection.

rhNRG-1β was injected into the tail vein within 24 hours after the firstinjection of Adriamycin, the injection of rhNRG-1 β was carried out oncedaily for 10 days. Dosage was adjusted according to the body weight,volume of the drug administered was 0.2 ml/100 g.

6.3 Method of the Experiment 6.3.1 Method of Construction of AnimalHeart Failure Model

Based on the <<Guidance Principle of New Drug Preclinical Experiment>>,3.3 mg/kg of Adriamycin was injected into the tail vein, once per weekfor consecutive 4 injections, establishing Adriamycin caused toxicmyocarditis rat model.

6.3.2 Pharmacodynamic Experiment

rhNRG-1 β was injected into the tail vein within 24 hours after thefirst injection of Adriamycin. During the experiment, animal survivalwas dynamically monitored, heart function indexes (maximal rate ofpressure increase within left ventricle, dt/dpmax, maximal rate ofpressure decrease within left ventricle, −dp/dt, systolic end pressureof left ventricle LVPmax, diastolic end pressure of left ventricleLVPmin) were monitored at the 5^(th) week. Section was made from theheart tissue and pathological changes examined.

6.3.3 Observation Index 6.3.3.1 Survival Rate

Situation of survival was recorded weekly and the survival rate of thevarious experimental groups was calculated. Survival rate (%)=survivalanimal number/number of experimental animals×100.

6.3.3.2 Ratio of Heart Weight/Body Weight, Pathologic Section of HeartTissue

Heart was extirpated after thoracotomy (auricle should be reserved), theheart weight was measured after dried with absorbent paper, ratio ofheart weight/body weight calculated; outer diameter of the heart wasmeasured at ½ site of the vertically standing heart; medium part of theleft ventricle was crosswise cut open, maximal thickness of free wall ofthe left ventricle measured; the heart was fixed with 10% formaldehyde,paraffin embedded and HE stained, observation of the myocardialstructure carried out under optic microscope, give out pathologic score.

Criteria of the Pathologic Scoring:

Grade 0: normal myocardial structure, without atrophy or hypertrophy ofthe myocardial cells, with vacuole, cross striation clear; regulararrangement of the myocardium; endocardium and pericardium withoutabnormality; no changes in blood vessel and interstitial tissue.Grade 1: Focal dissolve of myocardial cytoplasm and vacuole formationwere seen in sporadic individual myocardial cells, while the neighboringmyocardial cells still looked normal.Grade 2: Atrophy, dissolve of myocardial cytoplasm and vacuole formationwas seen in small to medium extent of clustering myocardial cells, smallfocal necrosis of myocardial cells was also seen.Grade 3: Large extent of diffuse atrophy of myocardial cells, dissolveof cytoplasm or vacuole formation with quite marked necrosis. Scoringcould be performed between grade 1 and grade 2 or between grade 2 andgrade 3, e.g. grade 1.5, grade 2.5 etc.

6.3.3.3 Determination of Hemodynamics Index

Hemodynamics index such as carotid artery pressure, intra-ventricularpressure. dp/dt was measured with six lead physiology recorder. Majorprocedure: separate the right carotid artery, ligate its distal end andblock its proximal end with arterial clamps, 20 G arteriovenousindwelling needle was inserted into the carotid artery, took out themedal stylet, loosened the artery clamps, push the plastic trocarfurther into appropriate depth, indwelled for 10 minutes, observed thewave style recorded by the physiology recorder, after it was stable,then recorded the carotid artery pressure and push the trocar furtherinto the left ventricle, kept in place for 15 minutes, after it wasstable, then recorded the dp/dt, −dp/dt, LVP_(max) and LVP_(min).

6.3.3.4 Determination of Serum Troponin T (cTnT) Level

2 ml of arterial blood was withdrawn, serum extracted, frozen and storedat −20° C., determination of serum cTnT content was performed withelectrochemistry irradiate method submitted to be carried out byClinical Laboratory of Zhong Shan Hospital.

7. Data Processing

Data was expressed as X±SD. Inter-group difference was analyzed withmonofactorial variance analysis.

8. Results of the Experiment

8.1 Adriamycin could Induce Toxic Myocarditis Complicated with HeartFailure in Rat

Refer to Table 10, and FIG. 18. 3.3 mg/kg of Adriamycin wasintravenously injected once every week for 4 consecutive injections, 5weeks later, animal survival rate was 15%, significant heart functiondamage was seen in the survived rat, their dp/dt, −dp/dt, LVPmax, LVPminwas 43%, 47%, 58% and 37% of the normal value respectively, pathologicscoring of the myocardial tissue was 2.33±0.26, relative morbidity ratewas 100%, serum cTnT raised significantly and reached 0.2 ng/ml,demonstrating that toxic myocarditis and the resulting heart failurecaused by Adriamycin animal model was successfully established.

TABLE 10 Various determination index of toxic myocarditis induced byAdriamycin rat model Control group Model animal group survival rate (%)100 15 pathologic score of  0 2.33 ± 0.26 * myocardial tissue dp/dt(mmHg/s) 6235 ± 423  2674 ± 446 ** −dp/dt −4590 ± 1003  −2141 ± 596 ** LVPmax (mmHg) 181.4 ± 15.4  106.1 ± 21.2 *  LVPmin −27.1 ± 10.2  −10.0 ±4. 7 **  cTnT (ng/ml) 0.001 ± 0.000  0.205 ± 0.072 ** heart weight/bodyweight 0.0032 ± 0.0002 0.0031 ± 0.0001  thickness of left 1.88 ± 0.151.73 ± 0.16  ventricular wall (mm) heart circumference (mm) 31.0 ± 1.1 30.6 ± 0.1   n = 20, X ± SD, when comparing with that of the normalgroup, variance analysis, * P < 0.05 ** P < 0.001.

FIG. 18 illustrates myocardium pathologic section of SD rat toxicmyocarditis induced by Adriamycin; a: normal control group: pathologicscore of myocardium was 0, without myocardial cells atrophy andhypertrophy, with vacuole formation, cross striation can be clearlyseen; myocardium arranged regularly; no abnormality of endocardium andpericardium; no changes of the vessels and interstitial tissue; b: modelanimal group: pathologic score of the myocardium was 3, large area ofmyocardial cell necrosis and dissolution.

8.2 Effect of rhNRG-1 β on Survival Rate of the Model Animals

The results showed that with time of the experiment extended, all of thethree-dosage levels of rhNRG-1 β could significantly reduce mortality ofthe model animals, survival rate of the model animal of 20 μg/kg dosagegroup reached up to 90% (P<0.01) (FIG. 19).

8.3 Effect of rhNRG-1 β on Heart Function of Model Animals

After the drug administration, dp/dt and LVPmax in all the testing druggroup animals raised somewhat. Through monofactorial variance analysiswith SPSS software, intergroups comparison showed that dp/dt and −dp/dtof testing drug group animals was significantly higher than those of themodel animal group (P<0.001), the difference was not significant whencomparing with those of the control group (P>0.05). Those of 40 and 20μg/kg dosage group were significantly higher than those of 10 μg/kggroup (P<0.01); LVPmax of the testing drug group (40, 20 and 10 μg/kg)was significantly higher than that of the model animal group (P<0.001)as well, in addition, the difference between various testing drug groupswas significant statistically (P<0.05), demonstrating that rhNRG-1 βcould effective improve heart function of the model animals and wasdose-dependent. Results of the two experiments were coincident (Tables11 and 12).

TABLE 11 Effect of rhNRG-1 β on heart function of model animals (I) Drugadminis- +dp/dt −dp/dt LVPmax LVPmin Group tration (mmHg/s) (mmHg/s)(mmHg) (mmHg) Normal con- 6235 ± −4590 ± 181.4 ± −27.1 ± trol group423** 1003** 15.4** 10.2** (n = 8) Model group iv 2674 ± −2141 ± 106.1 ±−10 ± (n = 6) qd × 10 d 446 596 21.2 4.7 rhNRG-1 β iv 5954 ± −4794 ±165.7 ± −27.4 ± 40 μg/kg qd × 10 d 689** 954** 22.7** 10** (n = 8)rhNRG-1 β iv 6107 ± −4323± 156.1 ± −26.9 ± 20 μg/kg qd × 10 d 418**457** 17.7** 9.7** (n = 8) rhNRG-1 β iv 4875 ± −3672 ± 145 ± −24.5 ± 10μg/kg qd × 10 d 636** 884** 15.2** 9.6** (n = 8) *p < 0.05; **p < 0.001,when comparing with that of the model animal group

TABLE 12 Effect of rhNRG-1 β on heart function of model animals (II)Drug adminis- +dp/dt −dp/dt LVPmax LVPmin Sample tration (mmHg/s)(mmHg/s) (mmHg) (mmHg) Normal 5872 ± −4626 ± 159 ± −22.7 ± group 342**896** 25** 12* (n = 8) Model iv 2675 ± −2137 ± 103.9 ± −11.3 ± animalgroup qd × 10 d 359 334 11.5 5.4 (n = 8) rhNRG-1 β iv 6041 ± −4529 ±166.3 ± −22.2 ± 40 μg/kg qd × 10 d 461** 274** 12.4** 11.4* (n = 7)rhNRG-1 β iv 5833 ± −4345 ± 157.7 ± −26.6 ± 20 μg/kg qd × 10 d 416**807** 12** 7.4* (n = 7) rhNRG-1 β iv 4956 ± −3626 ± 158.2 ± −22.4 ± 10μg/kg qd × 10 d 352** 1056** 22.9** 18 (n = 8) *p < 0.05; **p < 0.001,when comparing with that of the model animals8.4 Effect of rhNRG-1 β on Myocardial Structure of Model AnimalsrhNRG-1β could significant reduce the severity of myocardial cellsdamage in model animals, significant reduce pathologic score and thedifference was significant statistically when comparing with that of themodel group (P<0.01, P<0.05). Tables 13 and 14 and FIG. 20 showed theresults of the two experiments.FIG. 20. Effect of rhNRG-1 β on Myocardial Structure of Model Animals

a: model animal myocardium pathologic score was 3, large area ofmyocardial cells necrosis and dissolution; b: 40 μg/kg testing druggroup, myocardial pathologic score was 1, most of the myocardial cellswere normal, there was local myocardial cytoplasm dissolution insporadic myocardial cells; c: 20 μg/kg testing drug group, myocardialpathologic score was 1, most of the myocardial cells were normal, therewas local myocardial cytoplasm dissolution in sporadic myocardial cells;d: 130 μg/kg testing drug group, myocardial pathologic score was 1.5,vacuole degeneration was seen in small cluster of myocardial cells.

TABLE 13 Effect of rhNRG-1β on myocardial pathologic changes of modelanimals (I) Group Drug administration Pathologic scoring Normal group(n= 6) 0.00 ± 0.00** Model group (n = 6) iv qd × 10d 2.33 ± 0.26  rhNRG-1β40 μg/kg (n = 6) iv qd × 10d 0.33 ± 0.41** rhNRG-1β 20 μg/kg (n = 6) ivqd × 10d 1.17 ± 0.93*  rhNRG-1β 10 μg/kg (n = 6) iv qd × 10d 1.83 ±0.41*  *p < 0.05; **p < 0.01 when comparing with that of the modelanimal group

TABLE 14 Effect of rhNRG-1β on myocardial pathologic changes of modelanimals (II) Group Drug administration Pathologic scoring Normal group(n= 6)  0 ± 0** Model animal group (n = 6) iv qd × 10d 2.75 ± 0.274rhNRG-1β 40 μg/kg (n = 6) iv qd × 10  0.583 ± 0.204** rhNRG-1β 20 μg/kg(n = 6) iv qd × 10d 1.667 ± 0.931* rhNRG-1β 10 μg/kg (n = 6) iv qd × 10d2.167 ± 0.516* p < 0.05; **p < 0.01, when comparing with that of themodel animal group,8.5 Effect of rhNRG-1 β on Serum cTnT of Model Animals

After the drug administration, serum cTnT content reduced significantlyin every group animals, serum cTnT of high, medium and low dosage levelgroups was significant lower than that of the model group (P<0.001).Results of the two experiments were coincidence (Tables 15 and 16).

TABLE 15 Effect of rhNRG-1β on serum cTnT of model animals (I) GroupDrug administration cTnT (ng/ml) Normal group(n = 5) 0.001 ± 0.000**Model group (n = 6) iv qd × 10d 0.205 ± 0.072  rhNRG-1β 40 μg/kg (n = 6)iv qd × 10d 0.025 ± 0.011** rhNRG-1β 20 μg/kg (n = 6) iv qd × 10d 0.031± 0.006** rhNRG-1β 10 μg/kg (n = 6) iv qd × 10d 0.074 ± 0.024** **p <0.001 when comparing with that of the model group

TABLE 16 Effect of rhNRG-1β on serum cTnT of model animals (II) GroupDrug administration cTnI (ng/ml) Normal group(n = 6) 0.433 ± 0.079**Model group (n = 6) iv qd × 10d 20.525 ± 20.638  rhNRG-1β 40 μg/kg (n =6) iv qd × 10 0.874 ± 0.108** rhNRG-1β 20 μg/kg (n = 6) iv qd × 10d1.677 ± 0.589** rhNRG-1β 10 μg/kg(n = 6) iv qd × 10d  8.342 ± 13.537****p < 0.001 when comparing with that of the model animal group8.6 Effect of rhNRG-1 β on Heart Size of Model Animals

Table and 18 showed results of the two experiments. There was nosignificant change in heart physical parameters of the testing druggroups and the difference was not significant statistically between thegroups (P>0.05).

TABLE 17 Effect of rhNRG-1β on heart size of model animals (I) DrugHeart weight/ Left ventricle wall Group administration body weightthickness (mm) Normal 0.0032 ± 0.0002  2.01 ± 0.07** group (n = 20)Model iv qd × 10d 0.0031 ± 0.0001 1.717 ± 0.154 animal group (n = 6)rhNRG-1β iv qd × 10d 0.0031 ± 0.0002 1.813 ± 0.12  40 μg/kg (n = 16)rhNRG-1β iv qd × 10d 0.0032 ± 0.0001 1.789 ± 0.133 20 μg/kg (n = 18)rhNRG-1β iv qd × 10d 0.00301 ± 0.0002  1.773 ± 0.115 10 μg/kg (n = 13)**p < 0.01, when comparing with that of the model animal group

TABLE 18 Effect of rhNRG-1β on heart size of model animals (II) LeftDrug Heart weight/ ventricular wall Group administration body weightthickness (mm) Normal 0.00310 ± 0.000  2.19 ± 0.2** group(n = 20) Modelgroup iv qd × 10d 0.00298 ± 0.000 2.065 ± 0.17  (n = 10) rhNRG-1β iv qd× 10 0.00297 ± 0.000 2.06 ± 0.2  40 μg/kg (n = 18) rhNRG-1β iv qd × 10d0.00303 ± 0.000 2.15 ± 0.24 20 μg/kg (n = 19) rhNRG-1β iv qd × 10d0.00307 ± 0.000 2.18 ± 0.21 10 μg/kg g(n = 16) **p < 0.01, whencomparing with that of the model animal group

9. Conclusion

40, 20 and 10 μg/kg of rhNRG-1β could significantly improved thesurvival rate, reaching 85%, 90% and 60% respectively when comparingwith that of 15% survival rate of the model group; dp/dt, −dp/dt andLVPmax of the high, medium and low dosage level group were significantlyincreased, dp/dt was 5954±689, 6107±418, 4875±636 respectively, −dp/dtwas −4794±954, −4323±457, −3672±884 respectively, and LVPmax was165.7±22.7, 156.1±17.7, 145±15.2 respectively, there was significantdifference when comparing with that of the control group (P<0.001), inaddition, dp/dt, −dp/dt and LVPmax of the 40 and 20 μg/kg dosage groupdiffered significantly from those of the 10 μg/kg group (<0.05) and wassomewhat dose dependent; all the three-dosage levels of 40, 20 and 10μg/kg rhNRG-1 β could effectively alleviate the severity of myocardialdamage in the model animal, reduce serum troponin T (cTnT) content,being 0.025±0.011, 0.031±0.006 and 0.074±0.024 respectively; whencomparing with the 0.205±0.072 of the model animal group, there wassignificant difference. (P<0.01).

Results of the experiment showed that rhNRG-1 β could effectively treattoxic myocardial injury cause by Adriamycin in rat through reduced serumrelease of cTnT and myocardial fiber necrosis, improved contractionfunction of the heart and reduced animal mortality.

Example 4 Therapeutic Effect of rhNRG-1 β on Acute Myocardial InjuryCaused by Viral Infection in Mice 1. Abstract

Objectives To study therapeutic effect of rhNRG-1 β on acute myocardialinjury caused by viral (Coxsackie B3) infection. Method Mmice acuteviral myocarditis model was established through intra-abdominalinjection of Coxsackie B3 virus (CVB₃). The model animals were randomlydivided into groups, i.e., normal control group, model group, testingdrug group with 20 animals in each group. Three dosage level of 30, 15and 7.5 μg/kg of rhNRG-1β was established and injection into the tailvein was carried out at the same day, for consecutive 5 days. During theexperiment, animal survival rate was monitored. Heart function test(echocardiograph) was performed at the 7^(th) day and killed the animalsat the 8^(th) day, serum was extracted for troponin I (cTnI) leveldetermination and heart pathologic examination performed. Results EFvalue (90.2±2.5%, 86.0±2.9%) and FS value (55.7±2.1%, 50.7±4.3%) of boththe 30 μg/kg and 15 μg/kg group increased significantly; there wassignificant difference when comparing with those of the model group(P<0.01), LVDd value (0.187±0.006, 0.189±0.008) and LVDs value(0.085±0.009, 0.099±0.027) were significantly lower than those of themodel group (0.208±0.015, 0.142±0.020) (P<0.05); rhNRG-1 β couldalleviate the severity of myocardial pathologic injury, effectivelyreduced serum troponin (cTnI) level, cTnI of 30 μg/kg dosage group(7.98±6.07 ng/ml) and 15 μg/kg group (19.43±10.76 ng/ml) weresignificantly lower than that of the model group (44.44±12.39 ng/ml),the difference between them was significant statistically (P<0.001,P<0.005); 30 μg/kg of rhNRG-1β could significantly improved survivalrate of the model animals, reaching 80%, P<0.05. Conclusion Certaindosage of rhNRG-1 β could effective threat acute myocardial injurycaused by viral infection.

2. Objectives of the Experiment

To study therapeutic effect of rhNRG-1 β on acute myocardial injurycaused by viral infection in mice and to find out optional effectivedosage.

3. Testing Drug

rhNRG-1 β, provided by Zensun (Shanghai) Science & TechnologyDevelopment. Batch number: 200110006-2 Titer: 5000 u; purity:>95%(HPLC-C8).

4. Experiment Animal

-   4.1 Species, source and certificate of competency: 4-week old    purebred BALB/C mice, provided by Experiment Animal Department of    Fudan University, Number of certificate of animal competency: Yi    Dong Zi 22-9.-   4.2 Body weight and gender: 10-12 g, male.-   4.3 Animal number in each group: 20 animals in each experimental    group, 10 in the normal control group.

5. Virus

Coxsackie Virus B3, CVB₃, Nancy strain, provided by Ministerial ViralHeart Disease Laboratory (Shanghai Municipal Cardiovascular DiseasesInstitute).

6. Reagents and Equipment

-   6.1 Echocardiography device, Hewlett Packard sonos 5500; type of the    probe: S12′;-   6.2 Immuno-Assay System Opus® Plus, produced by Behring Diagnostic    Inc. for determining serum troponin I (cTnI), batch number: CTE8;-   6.3 Precise electronic balance, KERN 822;-   6.4 Water for injection, Zang Jiang Andus Bioproduct Co Ltd, 10×5    ml, batch number: 0112180;-   6.5 Epilating agent, 8% sodium sulfide, GuangDong XiLong Chemical    Plant, batch number: 010622.

7. Method of the Experiment 7.1 Experiment Grouping

Normal control group, model group, testing drug group and placebocontrol group were set up;Model group was negative control group (n=20): Prepared buffer solutionwas administered (10 mN PB, 0.2% human serum albumin, 5% mannitol);Testing drug group (n=20): high, medium and low dosage level of rhNRG-1βgroup were divided;Placebo control group (n=20): Intra-abdominal injection of non-CVB3freeze-thaw cellular supernatant, 0.2 ml/animal.

7.2 Dosage Set Up, Preparation of Testing Drug, Route of DrugAdministration, Times of Drug Administration, Concentration and Volumeof the Testing Drug

Three-dosage levels of 30, 15 and 7.5 μg/kg of the testing drug groupwere established based on the results of preliminary experiment. Dilutedwith preparation buffer solution (10 mM PB, 0.2% human serum albumin, 5%mannitol) to needed concentration.

Drug administration of both the testing drug group and the model groupwas intravenous injection into the tail vein of mice, once every day(qd), for consecutive 5 days, volume of each of the drug administeredwas 0.2 ml/animal.

7.3 Method of the Experiment 7.3.1 Set Up of Acute Viral MyocariditisAnimal Model in Mice

0.2 ml of 100×TCID₅₀ CVB₃ provided by Zhong Shan Hospital affiliated toFudan University was injected intra-abdominally and establishedmyocarditis model. Within the following week, the mouse manifestedpilo-erection, depilation, emaciation, dullness and death, about half ofthe tested animals died at the 8^(th) day.

7.3.2 Pharmacodynamic Experiment

Intravenous injection of the testing drug into the tail vein was carriedout the same day of intra-abdominal viral infection in mice, theinjection was performed for consecutive 5 days, animal survival rate wasmonitored, after completion of the experiment, echocardiograph,myocardial pathologic examination, serum troponin determination werecarried out.

7.3.3 Observation Index 7.3.3.1 Heart Function Measurement on Mice

Chest depilating was performed at the 7^(th) day of viral injection,then the mouse was fixed on special made fixed mount and echocardiographwas carried out with S12 high-frequency probe, the main index included:

-   EF: ejection fraction of left ventricle, being major index    reflecting ejection function of left ventricle;-   FS: shortening fraction of left ventricle, index reflecting    contraction function of left ventricle;-   LVDd: diastolic maximal inner diameter (cm);-   LVDs: systolic minimal inner diameter (cm).    7.3.3.2 Determination of Serum Troponin I (cTnI) in Mice

Severity of myocardial injury was evaluated through determination of theamount of serum cTnI release, with advantage of high specificity andhigh sensitivity when comparing with other traditional index such as CK,LDH and AST. Therefore, serum cTnI content determination was used as anobjective index reflecting the severity of myocardial injury.

At the 8^(th) day of viral injection, body weight of the mice wasmeasured and blood withdrawn from the orbit, serum separated, stored in−20° C. refrigerator and serum cTnI determination with luminous reactioncarried out.

7.3.3.3 Pathologic Examination of the Myocardium in Mice

The examination was submitted to be carried out by Pathology Departmentof Shanghai Medical College affiliated to Fudan University.

The survived mice were killed through dislocating the cervical spine,aseptic thoracotomy was performed and the heart extirpated, heart weightmeasured and put the myocardium into formaldehyde for fixation, embeddedin paraffin, consecutive section made and pathologic examinationperformed, observed inflammatory cells infiltration of the myocardium,degeneration and necrosis. Based on the Principle of New Drug EvaluationGuideline, pathologic scoring criteria was defined as follow:

Score 0: lesion area accounting for 0%;Score 1: lesion area accounting for 25%;Score 2: lesion area accounting for 50%;Score 3: lesion area accounting for 75%;Score 4: lesion area accounting for 100%;Scoring could be performed between score 1 and score 2 or between score2 and score 3, e.g. when lesion area was 80% was scored as score 3.2,

7.3.3.4 Observation on Survival of Mice

The death status of model animals in various testing drug group wasmonitored.

8. Data Processing

Pairing t test of the relevant data was used for data processing.

9. Results

9.1 Effect of rhNRG-1 β on Heart Function of Mice Infected with Virus

The results showed that both EF value (67.1±9.9%) and Fs value(32.0±7.2%) were significantly lower than the normal value (EF,92.5±2.3%/FS, 59.2±3.1%), p<0.05; EF value (90.2±2.5%, 86.0±2.9%) and FSvalue (55.7±2.1%, 50.7±4.3%) of 30 μg/kg and 15 μg/kg of rhNRG-1 β groupincreased significantly, there was significant difference when comparingwith those of the model group (P<0.01). EF/FS of 7.5 μg/kg group did nothave significantly difference from that of the model group (P>0.05).

LVDd value (0.208±0.015 cm) and LVDs value (0.142±0.020 cm) of the modelgroup were significantly higher than that of the normal control group(LVDd, 0.179±0.007 cm/LVDs, 0.073±0.006 cm) and there were significantdifference between them (P<0.01). LVDd and LVDs of 30 μg/kg and 15 μg/kgof rhNRG-1 β group were significantly lower than that of the model group(P<0.05), while that of 7.5 μg/kg differed insignificantly from that ofthe model group (P>0.05). Tables 19 and 20 showed results of the 2repeated experiments.

TABLE 19 Determination parameters of heart function after 5-day drugadministration in mice infected with virus (I) Drug administration LVDdLVDs EF FS Group regime (cm) (cm) (%) (%) Normal control group 0.179 ±0.007** 0.073 ± 0.006** 92.5 ± 2.3** 59.2 ± 3.1** Model group iv 0.208 ±0.015  0.142 ± 0.020  67.1 ± 9.9  32.0 ± 7.2  qd × 5 hNRG-1β 30 μg/kg iv0.187 ± 0.006** 0.085 ± 0.009** 90.2 ± 2.5** 55.7 ± 2.1** qd × 5rhNRG-1β iv 0.189 ± 0.008*  0.099 ± 0.027*  81.3 ± 6.28* 44.5 ± 8.27* 15μg/kg qd × 5 rhNRG-1β iv 0.191 ± 0.012  0.114 ± 0.028  78.1 ± 9.3  41.7± 9.6  7.5 μg/kg qd × 5 In each of the above-described data, each groupn = 6, through SPSS one-way anova analysis and compare with that of themodel group *P < 0.05 **P < 0.01

TABLE 20 Determination parameters of heart function after 5-day's drugadministration in mice infected with virus (II) Drug administration LVDdLVDs EF FS Group regime (cm) (cm) (%) (%) Normal control group 0.189 ±0.008** 0.069 ± 0.006** 94.5 ± 0.56** 63.7 ± 3.01** Model group iv 0.232± 0.023  0.159 ± 0.031  63.1 ± 18.47  30.3 ± 10.75  qd × 5 rhNRG-1β iv0.196 ± 0.011** 0.094 ± 0.011** 87.7 ± 3.76** 51.8 ± 5.24** 30 μg/kg qd× 5 rhNRG-1β iv 0.201 ± 0.011*  0.114 ± 0.018*  81.6 ± 4.59*  44.9 ±5.68*  15 μg/kg qd × 5 hNRG-1β iv 0.213 ± 0.009  0.133 ± 0.015  71.9 ±6.69  35.7 ± 5.60  7.5 μg/kg qd × 5 In each of the above-described data,each group n = 6, through SPSS one-way anova analysis and compare withthat of the model group *P < 0.05 **P < 0.019.2 Effect of rhNRG-1 β on Serum cTnI of Mice Infected by Virus

Results showed that cTnI of the model group (44.44±12.39 ng/ml) wassignificant higher than that of the control group (3.28±74.55 ng/ml),that of 30 μg/kg of rhNRG-1 β Group (7.98±6.07 ng/ml) was significantlylower than that of the model group (P<0.01); cTnI of 15 μg/kg of rhNRG-1β group (19.43±10.76 ng/ml) differed significantly from that of themodel group (P<0.05), whereas there was no significant differencestatistically between cTnI of the 7.5 μg/kg of rhNRG-1 β_(group)(29.05±17.06 ng/ml) and that of the model group. Tables 21 and 22 showedthe results of the two experiments.

TABLE 21 Effect of rhNRG-1β on serum cTnI (ng/ml) content in miceinfected by virus (I) Drug administration cTnI (ng/ml) Mean ± Groupregime SD Normal control group 3.28 ± 4.55** Model group iv 44.44 ±12.39  qd × 5 rhNRG-1β 30 μg/kg iv qd × 5 7.98 ± 6.07** rhNRG-1β 15μg/kg iv qd × 5 19.43 ± 10.76*  rhNRG-1β 7.5 μg/kg iv qd × 5 29.05 ±17.06  Placebo control group 3.75 ± 2.36** In each of theabove-described data, each group n = 6, through SPSS Nonparametric tests(Independent samples tests) analysis and compare with that of the modelgroup *P < 0.05 **P < 0.01

TABLE 22 Effect of rhNRG-1β on serum cTnI (ng/ml) content in miceinfected by virus (II) cTnI(ng/ml) Group Drug administration regime Mean± SD Normal group  0.19 ± 0.06** Model group iv 34.05 ± 16.50 qd × 5rhNRG-1β 30 μg/kg iv qd × 5  0.54 ± 0.53** rhNRG-1β 15 μg/kg iv qd × 5 15.59 ± 14.94* rhNRG-1β 7.5 μg/kg iv qd × 5 26.85 ± 15.20 Placebocontrol group    14 ± 0.03** In each of the above-described data, eachgroup n = 6, through SPSS Nonparametric tests (Independent samplestests) analysis and compare with that of the model group *P < 0.05 **P <0.019.3 Effect of rhNRG-1 β on Myocardial Injury of Mice Infected with Virus

Results of the experiment showed that myocardial pathologic score of thenormal group was 0.0±0.00, myocardial pathologic score of the modelgroup was (2.22±0.97), there was significant difference between them(P<0.01). Pathologic score of both the high dosage and medium dosagelevel of rhNRG-1 β decreased significantly (0.56±0.47 and 0.73±0.58respectively), and were significantly different from that of the modelgroup (P<0.001), there was no significant difference between that of thelow dosage level group and that of the model group (P>0.05). Tables 23and 24 and FIGS. 21 and 22 showed results of the two experiments.

TABLE 23 Effect of rhNRG-1β on myocardial pathologic changes in miceinfected by virus (I) Drug Pathologic score Group administration regimeMean ± SD Normal control group 0.00 ± 0.00** (n = 10) Model group (n =10) iv 2.22 ± 0.97  qd × 5 rhNRG-1β 30 μg/kg (n = 17) iv qd × 5 0.56 ±0.47** rhNRG-1β 15 μg/kg (n = 13) iv qd × 5 0.73 ± 0.58** rhNRG-1β 7.5μg/kg iv qd × 5 1.52 ± 0.74  (n = 10) Placebo control group 0.00 ±0.00** (n = 10) In each of the above-described data, each group n = 6,through SPSS Nonparametric tests (Independent samples tests) analysisand compare with that of the model group *P < 0.05 **P < 0.01

TABLE 24 Effect of rhNRG-1β on myocardial pathologic changes in miceinfected by virus (II) Pathologic score Group Drug administration regimeMean ± SD Normal control group 0.00 ± 0.00** (n = 10) Model group (n =10) iv 2.03 ± 1.44  qd × 5 rhNRG-1β 30 μg/kg iv qd × 5 0.23 ± 0.26** (n= 17) rhNRG-1β 15 μg/kg iv qd × 5 0.57 ± 0.58** (n = 13) rhNRG-1β 7.5μg/kg iv qd × 5 1.34 ± 1.16  (n = 10) Placebo control group 0.00 ±0.00** (n = 10) In each of the above-described data, each group n = 6,through SPSS Nonparametric tests (Independent samples tests) analysisand compare with that of the model group * P < 0.05 ** P < 0.019.4 Effect of rhNRG-1 β on Survival Rate of Mice Infected by Virus

Tables 25 and 26 showed results of the two experiments. One week afterinjection of the virus, survival rate of mice in the model group was 50%and 55% respectively, while after injection of rhNRG-1 β they raised upto 85% and 80% (30 μg/kg) and 70% and 65% (15 μg/kg).

TABLE 25 Effect of rhNRG-1 β on survival rate of mice infected by virus(I) Drug adminis- Numbers of survival animals (relative survival rate %)Group tration 1^(st) day 2^(nd) day 3^(rd) day 4^(th) day 5^(th) day6^(th) day 7^(th) day killed Normal control 10 10 10 10 10 10 10  10**group 100% 100% 100% 100% 100% 100%  100%  100%  (n = 10) Model group ivqd × 5 20 20 20 18 17 14 11 10 (n = 20) 100% 100% 100%  90%  85% 70% 55%50% rhNRG-1 β iv qd × 5 20 20 20 20 20 18 17  16* 30 μg/kg 100% 100%100% 100% 100% 90% 85% 80% (n = 20) rhNRG-1 β iv qd × 5 20 20 20 20 1917 14 13 15 μg/kg 100% 100% 100% 100%  95% 85% 70% 65% (n = 20) rhNRG-1β iv qd × 5 20 20 20 19 18 15 13 10 5 μg/kg 100% 100% 100%  95%  90% 75%65% 50% (n = 20) Placebo control 20 20 20 20 20 20 20  20** group 100%100% 100% 100% 100% 100%  100%  100%  (n = 20) The above described datawere processed through SPSS software, Survival Life Tables analysis, *P< 0.05 **P < 0.01, when comparing with that of the control group

TABLE 26 Effect of rhNRG-1 β on survival rate of mice infected by virus(II) Drug adminis- Numbers of survival animals (relative survival rate%) Group tration 1^(st) day 2^(nd) day 3^(rd) day 4^(th) day 5^(th) day6^(th) day 7^(th) day killed Normal control 10 10 10 10 10 10 10  10**group 100% 100% 100% 100% 100%  100%  100%  100%  (n = 10) Model groupiv qd × 5 20 20 20 18 15 13 10 10 (n = 20) 100% 100% 100%  90% 75% 65%50% 50% rhNRG-1 β iv qd × 5 20 20 20 20 19 18 17  17* 30 μg/kg 100% 100%100% 100% 95% 90% 85% 85% (n = 20) rhNRG-1 β iv qd × 5 20 20 20 19 18 1613 13 15 μg/kg 100% 100% 100%  95% 90% 80% 65% 65% (n = 20) rhNRG-1 β ivqd × 5 20 20 20 18 17 14 11 10 7.5 μg/kg 100% 100% 100%  90% 85% 70% 55%50% (n = 20) Placebo control 20 20 20 20 20 20 20  20** group 100% 100%100% 100% 100%  100%  100%  100%  (n = 20) The above described data wereprocessed through SPSS software, Survival Life Tables analysis, *P <0.05 **P < 0.01 when comparing with that of the control group

9. Conclusion

EF value (90.2±2.5%, 86.0±2.9%) and FS value (55.7±2.1%, 50.7±4.3%) of30 μg/kg and 15 μg/kg of rhNRG-1 β groups raised significantly and therewas significant difference when comparing with that of the model group(P<0.02), both LVDd value (0.187±0.006, 0.189±0.008) and LVDs value(0.085±0.009, 0.099±0.027) were lower than that of the model group(0.208±0.015, 0.142±0.020), P<0.05; rhNRG-1 β could alleviate theseverity of myocardial pathologic damage of model group animals,effectively reduced serum troponin I (cTnI) level, cTnI content of 30μg/kg dosage level group (7.98±6.07 ng/ml) and 15 μg/kg dosage levelgroup (19.43±10.76 ng/ml) were significantly lower than that of themodel group (44.44±12.39 ng/ml) and there was significant differencebetween them (P<0.001, P<0.005); 30 μg/kg of rhNRG-1 β couldsignificantly improve survival rate of model group animals and reached80% when comparing with the 50% survival rate of the model groupanimals, P<0.05.

Results of the experiment showed that 30 μg/kg dosage level of rhNRG-1 βcould effectively treat acute myocardial injury caused by viralinfection in mice.

Example 5 Observation on Therapeutic Effect of rhNRG-1 β on AcuteMyocardial Injury of Mice Caused by Viral Infection (II) 1. Abstract

Objectives To study the effective time of drug administration of rhNRG-1β in the treatment of acute myocardial injury in mice caused by viralinfection. Method Acute myocardial injury animal model was establishedthrough intra-abdominal injection of Coxsacki B₃ virus (CVB₃) in mice.The model animals were randomly divided into groups, i.e., normalcontrol group, model group, testing drug group, 20 animals was assignedto each group. 30 μg/kg of rhNRG-1 β was injected into the tail vein forconsecutive 3, 5 and 7 days respectively. During the experiment, animalsurvival rate was monitored, echocardiograph was performed at the 7^(th)day and the animals were killed at the 8^(th) day, serum separated forcTnI level determination, heart pathohistological examination carriedout. Results Consecutive injection of 30 μg/kg of rhNRG-1 β for 3, 5 and7 days could raise the EF/FS value and there was significant difference(P<0.001) in comparing the EF/FS value of 5-day and 7-day testing druggroups (86.8±4.4%/51.9±5.8%, 87.0±3.3%/51.8±5.1%) with the EF/FS valueof the model group (66.5±5.6/31.8±3.7), the LVDs value of the 5 and7-day testing drug group decreased significantly (being 0.090±0.011,0.092±0.012 cm respectively) and there was significant difference(P<0.01) when comparing with that of the model group (0.133±0.012);rhNRG-1 β could alleviate the severity of myocardial pathologic damage,effectively reduced serum troponin I (eTnI) level of the model animals,the cTnI of 5-day testing drug group (1.06±1.32 ng/ml) and of 7-daytesting drug group (1.05±1.2 ng/ml) was significantly lower than that ofthe model group (23.54±16.96 ng/ml) P<0.01; consecutive 5 and 7-dayinjection of rhNRG-1 β could significantly improved the animal survivalrate, reaching 85% when comparing with the 50% survival rate of themodel animals, P<0.05. Conclusion 30 μg/kg of rhNRG-1 β injected forconsecutive 5 days could effectively treat the acute myocardial injuryin mice caused by viral infection.

2. Objectives

To make clear the effective time of drug administration of rhNRG-1 β inthe treatment of mice acute myocardial injury caused by viral infection.

3. Testing Drug

rhNRG-1 β, provided by Zensun (Shanghai) Science & TechnologyDevelopment. Batch number: 200110006-2, Titer: 500 u; purity:>95%(HPLC-C8).

5. Experiment Animal

-   5.1 Species, source and certificate of competency: 4-week old    purebred BALB/C mice, provided by Experiment Animal Department of    Fudan University, Number of certificate of animal competency: Yi    Dong Zi 22-9.-   5.2 Body weight and gender: 13-15 g, male.-   5.3 Animal number in each group: 20 animals in each experimental    group, 10 in the normal control group.

5. Virus

The same as that described in the previous section.

6. Reagents and Equipment

The same as that described in the previous section.

10. Method of the Experiment 10.1 Experiment Grouping

Normal mice control group, model group and testing drug group were setup;Normal mice control group (n=10);Model group (n=20): Prepared buffer solution was injected;Testing drug group (n=20): 30 μg/kg of rhNRG-1 β was administered for 3,5 and 7 consecutive days as three difference therapeutic courses with 20animals assigned for each group;Placebo control group (n=20): Intra-abdominal injection of non-CVB3freeze-thaw cellular supernatant, 0.2 ml/animal.

10.2 Dosage Set Up, Preparation of Testing Drug, Route of DrugAdministration, Times of Drug Administration, Concentration and Volumeof the Testing Drug

rhNRG-1β was diluted with preparation buffer solution (10 mM PB, 0.2%human serum albumin, 5% mannitol to needed concentration;Three drug administration groups were set up, intravenous injection forconsecutive 3-day, 5-day and 7-day, drug volume for each dose was 0.2ml/animal;Drug administration of the model group was intravenous injection intothe tail vein of mice, once every day (qd), for consecutive 7 days, drugvolume for each dose was 0.2 ml/animal.

7.3 Method of the Experiment 7.3.1 Set Up of Acute Viral MyocariditisAnimal Model in Mice

0.2 ml of 100×TCID₅₀ CVB₃ provided by Zhong Shan Hospital affiliated toFudan University was injected intra-abdominally and establishedmyocarditis model. Within the following week the mouse manifestedpilo-erection, depilation, emaciation, dullness and death, about half ofthe tested animals died at the 8^(th) day.

7.3.2 Pharmacodynamic Experiment

The same as that described in the previous section.

7.3.3 Observation Index

The same as that described in the previous section.

8. Data Processing

Pairing t test of the relevant data was used for the data processing.

9. Results

9.1 Effect of rhNRG-1 β Administered for 3, 5 and 7 Days on HeartFunction of Mice Infected by Virus

Results showed that EF/Fs value (66.5±5.6%/31.8±3.7%) was significantlylower than that of the normal group (93.5±0.9%/68.1±1.3%), thedifference was significant statistically (P<0.01). EF/FS value of 5 and7 consecutive-day of rhNRG-1 β injection (86.8±4.4%/51.9±5.8%,87.0±3.3%/51.8±5.1%) differed significantly from that of the model group(P<0.001).

EF value of 3 consecutive days of rhNRG-1 β injection raised again(73.1±6.6%), however without significant difference when comparing withthat of the model group (P<0.05).

LVDs value (0.133±0.012 cm) was higher than that of the normal group(0.059±0.006 cm) and there was significant difference between them(P<0.01). LVDs value of rhNRG-1 β administered for 5 days and for 7 daysgroup was significantly reduced (0.090±0.011, 0.092±0.012 cmrespectively), and differed significantly from that of the model group(P<0.001). LVDs value of rhNRG-1 β administered for 3-day group(0.123±0.012 cm) differed insignificantly from that of the model group(P<0.05). Table-27 and 17-28 showed results of the two experiments.

TABLE 27 Effect of rhNRG-1β administered for different number of days onheart function of mice infected by virus (I) Drug administration Groupregime LVDd (cm) LVDs (cm) EF (%) FS (%) Normal control 0.179* ± 0.007 0.059** ± 0.006 93.5** ± 0.9 68.1** ± 1.3 group Model group iv qd × 70.194 ± 0.012  0.133 ± 0.012  66.5 ± 5.6  31.8 ± 3.7 rhNRG-1β iv qd × 30.194 ± 0.008  0.123 ± 0.012  73.1 ± 6.6  36.7 ± 4.7 30 μg/kg rhNRG-1βiv qd × 5 0.187 ± 0.006 0.090** ± 0.011 86.8** ± 4.4 51.9** ± 5.8 30μg/kg rhNRG-1β iv qd × 7 0.192 ± 0.008 0.092** ± 0.012 87.0** ± 3.351.8** ± 5.1 30 μg/kg In each of the above-described data, each group n= 6, through SPSS one-way anova analysis and compare with that of themodel group *P < 0.05 **P < 0.01

TABLE 28 Effect of rhNRG-1β administered for different number of days onheart function of mice infected by virus (II) Drug administration Groupregime LVDd (cm) LVDs (cm) EF (%) FS (%) Normal control 0.189** ± 0.008 0.080** ± 0.007 90.9** ± 2.6  57.5** ± 3.5  group Model group iv qd × 70.206 ± 0.008  0.126 ± 0.006 75.4 ± 5.2 39.0 ± 4.3 rhNRG-1β iv qd × 30.211 ± 0.016  0.121 ± 0.016 81.0 ± 5.4 43.9 ± 5.1 30 μg/kg rhNRG-1β ivqd × 5 0.199 ± 0.000  0.100* ± 0.014 85.8** ± 4.205 50.0** ± 6.350 30μg/kg rhNRG-1β iv qd × 7 0.194 ± 0.017 0.092** ± 0.008 87.283** ± 1.694 52.367** ± 1.847  30 μg/kg In each of the above-described data, eachgroup n = 6, through SPSS one-way anova analysis and compare with thatof the model group *P < 0.05 **P < 0.019.2 Effect of rhNRG-1 β on Serum cTnI of Mice Infected by Virus

Results showed that mean of cTnI of the normal group (0.12±0.03 ng/ml)and mean of cTnI of the model group raised significantly (2154±16.96ng/ml) and there was significant difference between them (P<0.001). cTnIvalue of rhNRG-1 β administered for both 5-day and 7-day group reducedsignificantly (being 1.06±1.32 ng/ml, 1.05±1.20 ng/ml respectively), anddiffered significantly from that of the model group (P<0.001), thedifference was insignificant between the 3-day drug administration groupand the model group (P>0.05). Tables 29 and 30 showed the results of thetwo experiments.

TABLE 29 Effect of rhNRG-1β on serum cTnI (ng/ml) in mice infected byvirus (I) cTnI (ng/ml) Group n = 9) Drug administration regime Mean ± SDNormal control group 0.12 ± 0.03** Model group iv qd × 7 23.54 ± 16.96 rhNRG-1β 30 μg/kg iv qd × 3 13.37 ± 9.53   rhNRG-1β 30 μg/kg iv qd × 51.06 ± 1.32** rhNRG-1β 30 μg/kg iv qd × 7 1.05 ± 1.20** Each of theabove group was analyzed by Nonparametric tests (Independent samplestests) of SPSS software, when comparing with that of the model group, *P< 0.05 **P < 0.01

TABLE 30 Effect of rhNRG-1β on serum cTnI (ng/ml) in mice infected byvirus (II) Drug administration cTnI (ng/ml) Group n = 8) regime Mean ±SD Normal control group 0.15 ± 0.03** Model group iv qd × 7 30.13 ±21.75  rhNRG-1β iv qd × 3 12.32 ± 18.36  30 μg/kg rhNRG-1β iv qd × 50.44 ± 0.24** 30 μg/kg rhNRG-1β iv qd × 7 0.51 ± 0.28** 30 μg/kg Each ofthe above group was analyzed by Nonparametric tests (Independent samplestests) of SPSS software, when comparing with that of the model group, *P< 0.05 **P < 0.019.3 Effect of rhNRG-1 β Administered for Different Number of Days onMyocardial Pathologic Injury of Mice Infected with Virus

Results of the experiment showed that pathologic score of the normalgroup was 0.0±0.00, pathologic score of the model group was increased(1.44±1.19), there was significant difference between them (P<0.01),pathologic score of rhNRG-1 β administered for 5-day and 7-day groupsreduced significantly (being 0.11±0.14 and 0.13±0.13 respectively) anddiffered significantly from that of the model group (P<0.01). There wasimprovement of myocardial injury of the 3-day drug administration groupas well (0.33±0.155) and there was significant difference when comparingwith that of the model group, however, improvement of the myocardialcells of the 5-day drug administration group was markedly better thanthat of the 3-day drug administration group and there was significantdifference between them (P<0.01), while there was no significantdifference in pathologic scoring between 5-day and 7-day drugadministration group.

Tables 31, 32 and FIGS. 21 and 24 showed results of the two experiments.

TABLE 31 Effect of rhNRG-1β administered for different number of days onmyocardial pathologic changes in mice infected by virus (I) DrugPathologic score Group administration regime Mean ± SD Normal controlgroup 0.00 ± 0.00**  Model group iv qd × 7 1.44 ± 1.19   rhNRG-1β 30μg/kg iv qd × 3 0.33 ± 0.155*  rhNRG-1β 30 μg/kg iv qd × 5 0.11 ±0.140** rhNRG-1β 30 μg/kg iv qd × 7 0.13 ± 0.132** Each of the abovegroup was analyzed by Nonparametric tests (Independent samples tests) ofSPSS software, when comparing with that of the model group, *P < 0.05**P < 0.01

TABLE 32 Effect of rhNRG-1β administered for different number of days onmyocardial pathologic changes in mice infected by virus (II) Pathologicscore Group (n = 10) Drug administration regime Mean ± SD Normal control0.00 ± 0.00**  group Model group iv qd × 7 1.86 ± 1.20   rhNRG-1β 30μg/kg iv qd × 3 0.55 ± 0.476*  rhNRG-1β 30 μg/kg iv qd × 5 0.17 ±0.157** rhNRG-1β 30 μg/kg iv qd × 7 0.19 ± 0.168** Each of the abovegroup was analyzed by Nonparametric tests (Independent samples tests) ofSPSS software, when comparing with that of the model group, *P < 0.05**P < 0.019.4 Effect of rhNRG-1 β on Survival Rate of Mice Infected by Virus

Tables 33 and 34 showed results of the two experiments, survival rate ofmice in the model group was 50% and 55% respectively, while that ofrhNRG-1 β administered for both 5-day and 7-day group raised up to 85%and 80% and there was significant difference when comparing with that ofthe model group, P<0.05. Survival rate of the 3-day group raised to 65%and 75%, however, there was no significant difference when comparingwith that of the model group (P<0.05).

TABLE 33 Effect of rhNRG-1 β administered for different number of dayson survival rate in mice infected by virus (I) Drug adminis- Number ofsurvival animal (relative survival rate %) Group tration 1^(st) day2^(nd) day 3^(rd) day 4^(th) day 5^(th) day 6^(th) day 7^(th) day killedNormal control 10 10 10 10 10 10 10  10** group 100% 100% 100% 100%100%  100%  100%  100%  n = 10 Model group iv qd × 7 20 20 20 18 16 1310 10 n = 20 100% 100% 100%  90% 80% 65% 50% 50% rhNRG-1 β Iv qd × 3 2020 20 19 18 16 14 13 30 μg/kg 100% 100% 100%  95% 90% 80% 70% 65% n = 20rhNRG-1 β iv qd × 5 20 20 20 20 19 19 18  17* 30 μg/kg 100% 100% 100%100% 95% 95% 90% 85% n = 20 rhNRG-1 β iv qd × 7 20 20 20 20 20 19 19 17* 30 μg/kg 100% 100% 100% 100% 100%  90% 90% 85% n = 20 Each of theabove group was analyzed by Survival Life Tables of SPSS software, whencomparing with that of the model group, *P < 0.05 **P < 0.01

TABLE 34 Effect of rhNRG-1 β administered for different number of dayson survival rate in mice infected by virus (II) Drug adminis- Number ofsurvival animals (relative survival rate %) Group tration 1^(st) day2^(nd) day 3^(rd) day 4^(th) day 5^(th) day 6^(th) day 7^(th) day killedNormal group 10 10 10 10 10 10 10  10** n = 10 100% 100% 100% 100% 100% 100%  100%  100%  Model group iv qd × 7 20 20 20 18 17 14 12 12 n = 20100% 100% 100%  90% 85% 70% 60% 60% rhNRG-1 β iv qd × 3 20 20 20 20 1916 15 15 30 μg/kg 100% 100% 100% 100% 95% 80% 75% 75% n = 20 rhNRG-1 βiv qd × 5 20 20 20 20 19 19 18  18* 30 μg/kg 100% 100% 100% 100% 95% 95%90% 90% n = 20 rhNRG-1 β iv qd × 7 20 20 20 20 20 19 18  18* 30 μg/kg100% 100% 100% 100% 100%  95% 90% 90% n = 20 Each of the above group wasanalyzed by Survival Life Tables of SPSS software, when comparing withthat of the model group, *P < 0.05 **P < 0.01

10 Conclusion

30 μg/kg of rhNRG-1 β intravenously injected for consecutive 3, 5 and 7days could all raise the EF/FS value, EF/FS value of drug administrationfor 5 and 7 days groups (86.8±4.4%/51.9±5.8%, 87.0±3.3%/51.8±5.1%)differed significantly from EF/FS value of the model group(66.5±5.6/31.8±3.7) (P<0.01), LVDs of both the 5 and 7 days groupsreduced significantly (being 0.090±0.011 and 0.092±0.012 cmrespectively) and differed significantly from that of the model group(0.133±0.012) (P<0.01); rhNRG-1 β could alleviate the severity ofmyocardial pathologic damage in model animals, effectively reduced serumtroponin I (cTnI) level, cTnI of drug administration for 5-day group(1.06±1.32 ng/ml) and for 7-day group (1.05±1.2 ng/ml) weresignificantly lower than that of the model group (23.54±16.96 ng/ml),(P<0.01); rhNRG-1 β intravenously injected for consecutive 5 days and 7days could significant raised survival rate of the model animals,reaching 85%. P<0.05.

Results of the experiment demonstrated that 30 μg/kg of rhNRG-1 βadministered for consecutive 5 days could effectively treat the acutemyocardial injury in mice infected by virus.

Example 6 Therapeutic Effect of rhNRG-1 β on Congestive Heart FailureCaused by Inferior Vena Cava Constriction 1. Abstract

Objectives To study therapeutic effect of rhNRG-1 β on congestive heartfailure caused by inferior vena cava constriction in dog. Methods Afterabout 1 week of constriction of inferior vena cava by 50%, EF valuereduced by about 20% or cardiac output reduced by 20% determined byechocardiograph, demonstrating that stable low output, congestive heartfailure animal model was established. Then randomly divided the animalsinto groups with 6 dogs for each group, three dosage level of rhNRG-1 β,i.e., 1, 3 and 10 μg/kg, was intravenously injected daily forconsecutive 5 days. Heart function (echocardiography) determination wasperformed after the drug administration; various hemodynamic parametersin model animals were analyzed through cervical vein and carotid arterycatheterization respectively. Results All of the three dosage level ofrhNRG-1β (1, 3 and 10 μg/kg) administrated for consecutive 5 days couldraise EF/FS value and cardiac output (CO) of model animals, there weresignificant difference between those value prior to and after the drugadministration and those of the model group (P<0.05, P<0.01); 1, 3 and10 μg/kg of rhNRG-1 β could effectively raise dp/dt of left ventricle inmodel animals, there was significant difference when comparing with thatof the model group (P<0.01), could effectively raised LVPmax of themodel animals, reduced LVPmin value and P<0.04 when comparing with thoseof the model group, whereas effect on the right ventricle was notapparent. Conclusion rhNRG-1 β could effectively treat congestive heartfailure caused by constriction of the inferior vena cava in dog.

2. Objectives

To demonstrate therapeutic effect of rhNRG-1 β on congestive heartfailure caused by constriction of the inferior vena cava in dog.

3. Testing Sample

rhNRG-1 β, provided by Zensun (Shanghai) Science & TechnologyDevelopment.

Batch number: 200110006-2; Concentration: 500 μg/ampule; Titer: 5000u/ampule;

Purity:>95% (HPLC-C8). 6. Experiment Animal

-   6.1 Species, source and certificate of competency: crossbred dog,    provided by Zhong Shan Hospital of Fudan University, being eligible    guarantied by Experiment Department of Fudan University.-   6.2 Body weight and gender: 13-18 kg, male.-   6.3 Animal number in each group: 6 animals in each experimental    group.

5. Materials and Equipment

-   5.1 Echocardiography device, Hewlett Packard sonos 5500; type of the    probe: S4-   5.2 Water for injection, Zang Jiang Antus Bioproduct Co Ltd, 10×5    ml, batch number: 0112180;-   5.3 High-frequency electric knife, Shanghai Hu Tong electronic    equipment factory, GD350-D;-   5.4 Electrocardiography recorder, Nihon Kohden ECG-6511;-   5.5 Monitoring electrode, Ludlow Company of Canada, model: MT-200;-   5.6 Physiology recorder, Equipment Research Center of Shanghai    Medical University, SMUP-B;-   5.7 Electric ventilator, Shanghai No. 4 Medical Equipment Factory;-   5.8 Trifid balloon floating catheter, Edwards 114F7.

6 Method of the Experiment 6.1 Experiment Grouping

Pseudo-operation group, model group and testing drug group were set up.

Pseudo-operation group (n=6): only thoracotomy but without constrictionof the inferior vena cava were carried out.

Model group (n=6): Prepared buffer solution was injected after theestablishment of heart failure model.

Testing drug group: rhNRG-1 β was injected after the establishment ofheart failure model.

6.2 Dosage Set Up, Preparation of Testing Drug, Drug AdministrationRegime

High, medium and low dosage level of 1, 3 and 10 μg/kg respectively ofrhNRG-1 β was diluted with preparation buffer solution (vehicle) toneeded concentration, intravenous injection once everyday forconsecutive 5 days.

Preparation buffer solution was injected intravenously once daily forconsecutive 5 days for the model group.

Volume of the drug administered was 0.8 ml/kg body weight.

6.3 Method of the Experiment 6.3.1 Set Up of Heart Failure Caused byConstriction of Inferior Vena Cava Animal Model in Dog

3% pentobarbital sodium (30 mg/kg) was injected into the peripheral veinto anesthetize the dog, then trachea intubated. Aseptic thoracotomizedvia right chest between the 4 and 5 rib, measured circumference of theinferior vena cava at 3 cm from the right auricle. Selected hard spoolwith circumference equal to ⅓−½ of that of the inferior vena cava,tighted the spool and the inferior vena cava together with #7 silkthread, drew out the spool, stopped all the bleeding, closed the chest.Bred for 1 week, echocardiography performed according to the amount ofascites, when EF reduced by about 20% or dilatation of the left heart byabout 20%, stable low cardiac output congestive heart failure animalmodel was established. Then intravenous injection of the drug wascarried out for consecutive 5 days. Thoracotomy without constricting theinferior vena cava was carried out for the pseudo-operation group.

6.3.2 Pharmacodynamic Experiment

After identifying the establishment of animal model, experiment wascarried out according to the animal grouping and drug administrationregime.

6.3.3 Observation Index

Heart function index determination was carried out under anesthesia ofthe dog prior to the operation, prior to and 5-day after the drugadministration. Major index included:

EF (Ejection Fraction): heart ejection fraction, i.e., ratio ofdifference between end diastolic volume of the ventricle (EDV) and endsystolic volume of ventricle (ESV) and end diastolic volume ofventricle, being general index used for reflecting pumping function ofventricle; especially reflecting the systolic function;FS: ventricular short axis shortening rate, being index reflectingcontraction function of ventricle,CO: cardiac output, i.e. blood volume ejected by the heart per minute.

6.3.3.2 Hemodynamic Index Determination

7F trifid balloon floating catheter was inserted into the right jugularvein, right auricular pressure, right ventricular pressure, pulmonarypressure and pulmonary wedge pressure were recorded.

6F trifid balloon floating catheter was again inserted into left carotidartery, aorta pressure and left ventricle pressure were recorded withphysiology recorder with the following major index: LVPmax, LVPmin,+dp/dt, and −dp/dt.

7. Data Processing

Paring t test or nonparameter test of the collected data with SPSSsoftware was carried out

8. Results

8.1 Effect of rhNRG-1 β on Heart Function of TIVCC Dog8.1.1 Effect of rhNRG-1 β on Left Ventricle EF/FS Value of TIVCC Dog

EF and FS value prior to the constriction in model animals were82.3±1.6% and 49.2±2.6% respectively through echocardiographyexamination and reduced to 59.1±7.3% and 29.3±3.9% respectively afterthe operation, there was significant difference between that prior toand that after the constriction (P<0.01, P<0.05), EF and FS valuecontinuously maintained at 55.5±10.9% and 28.5±6.6% level 5 days later,demonstrating that through constriction of the inferior vena cava, thecongestive heart failure dog model was established and was relativelystable.

EF and FS value of all the three dosage level groups after rhNRG-1 βinjection raised significantly, EF and FS of the low dosage level group(1 μg/kg) increased from 57.7±10.9 and 30.6±8.0 to 70.4±8.4 and39.7±5.7, there was significant difference between prior to and afterthe drug administration (P<0.05), meanwhile, there was significantdifference when comparing with that of the model group (P<0.05). Inaddition, EF/FS value of the medium and high dosage level group modelanimals increased significantly as well and there was significantdifference when compared that prior to and after the drug administrationwith that of the model group (P<0.01), Table 35 showed the results.

TABLE 35 Effect of rhNRG-1 β on EF/FS of the left ventricle in TIVCC dogFS (%) EF (%) Prior to drug After drug Prior to drug After drug Grouppreoperation administration administration Preoperation administrationadministration Pseudo- 51.7 ± 2.2  47.4 ± 1.3** 50.3 ± 2.2**   84.1 ±1.4  81.4 ± 1.1** 83.1 ± 1.6**   operation group Model group 49.2 ± 2.629.3 ± 3.9 28.5 ± 6.6    82.3 ± 1.6 59.1 ± 7.3  55.5 ± 10.9     rhNRG-1β 50.5 ± 3.3 27.7 ± 5.6 41.5 ± 3.1**^(▴▴) 82.9 ± 2.6 55.8 ± 10.0 74.7 ±3.2**^(▴▴) 10 μg/kg rhNRG-1 β 51.7 ± 2.9 29.9 ± 6.4 42.4 ± 4.4**^(▴▴)84.7 ± 2.8 58.0 ± 8.3  74.7 ± 4.6**^(▴▴) 3 μg/kg rhNRG-1 β 50.8 ± 4.030.6 ± 8.0 39.7 ± 5.7*^(▴ )  82.8 ± 3.0 57.7 ± 10.9 70.4 ± 8.4*^(▴ )  1μg/kg All of the above described groups, n = 6 *P < 0.05, when comparingwith that of the model group: **P < 0.01, when comparing with that ofthe model group; ^(▴)P < 0.05, when comparing that prior to and thatafter the drug administration ^(▴▴)P < 0.01, when comparing that priorto and that after the drug administration8.1.2 Effect of rhNRG-1 β on Cardiac Output (CO) of TIVCC Dog

Table 36 showed that rhNRG-1 β could significantly increase cardiacoutput of the model animals, cardiac output of the 1 μg/kg of rhNRG-1 βgroup increased from 2.4±0.5 to 3.7±0.8 and there was significantdifference when compared that prior to and that after the drugadministration (P<0.05), meanwhile, there was significant difference aswell when comparing with that of the model animals (P<0.05). CO changesof 3 and 10 μg/kg dosage level group was even significant (P<0.01).Results of heart rate changes determination showed that rhNRG-1 β hasless effect on heart rate. (no details were revealed).

TABLE 36 Effect of rhNRG-1β on cardiac output of TIVCC dog (Lmin) CO(L/min) Prior to drug After drug Group preoperation administrationadministration Pseudo-operation 4.3 ± 0.7 3.9 ± 0.6 4.0 ± 0.6** groupModel group 4.7 ± 1.3 2.5 ± 0.8 2.7 ± 0.5 rhNRG-1β 10 μg/kg 4.3 ± 0.61.9 ± 0.3 3.6 ± 0.7**▴▴ rhNRG-1β 3 μg/kg 4.3 ± 0.8 2.1 ± 0.7 4.0 ±0.9*▴▴ rhNRG-1β 1 μg/kg 4.2 ± 0.6 2.4 ± 0.5 3.7 ± 0.8*▴ All of the abovedescribed groups, n = 6 *P < 0.05, when comparing with that of the modelgroup; **P < 0.01, when comparing with that of the model group;; ▴P <0.05, when comparing that prior to and that after the drugadministration ▴▴P < 0.01, when comparing that prior to with that afterthe drug administration8.2 Effect of rhNRG-1 β on Blood Flow Dynamics of TIVCC Dog

Changes of left and right ventricular dp/dt and end systolicpressure/end diastolic pressure were determined with floatingcatheterization technique. The results showed that 1, 3 and 10 μg/kg ofrhNRG-1 β could effectively increased the left ventricular dp/dt ofanimals, there was significant difference when comparing with that ofthe model group (P<0.05); effectively raised the LVPmax, reduced LVPminvalue, when comparing with that of the model group, P<0.05; rhNRG-1 βhas less effect on left heart dp/dt, right ventricular +dp/dt andventricular end pressure, Tables 37 and 38 showed the results indetails.

TABLE 37 Effect of rhNRG-1β on left ventricular dp/dt of TIVCC dog GroupL(+dp/dt) (mmHg/s) L(−dp/dt) (mmHg/s) Pseudo-operation 5088.99 ±982.87** −3233.39 ± 923.82 group Model group 2017.75 ± 295.25 −2384.94 ±1062.31 rhNRG-1β 10 μg/kg 5104.88 ± 1332.05** −3658.34 ± 1390.97rhNRG-1β 3 μg/kg 5000.45 ± 1535.88** −3249.52 ± 973.32 rhNRG-1β 1 μg/kg  4024 ± 1006 635.63**   −2933 ± 613.44 All the above described groups n= 6 *P < 0.05, when comparing with that of the model group; **P < 0.01,when comparing with that of the model group;

TABLE 38 Effect of rhNRG-1β on left ventricular LVPmax/LVPmin of TIVCCdog Group LVPmax (mmHg) LVPmin (mmHg) Pseudo-operation 145.04 ± 15.17**−0.03 ± 6.48 group Model group  95.07 ± 11.62  2.42 ± 2.86 rhNRG-1β 10μg/kg 122.87 ± 17.37* −0.69 ± 1.05* rhNRG-1β 3 μg/kg 114.68 ± 17.12*−1.12 ± 1.34* rhNRG-1β 1 μg/kg 102.12 ± 12.42  0.59 ± 3.05 All the abovedescribed groups n = 6 *P < 0.05, when comparing with that of the modelgroup; **P < 0.01, when comparing with that of the model group;

10 Conclusion

All the three dosage level (1, 3 and 10 μg/kg) of rhNRG-1 β administeredfor consecutive 5 days could raise the EF/FS value and cardiac output(CO) of the model animal, there was significant difference whencomparing that prior to with that after the drug administration(P<0.005, P<0.01); 1, 3 and 10 μg/kg of rhNRG-1β could effectivelyincrease left ventricular dp/dt and there was significant differencewhen comparing with that of the model group (P<0.05), being able toeffectively raise the LVPmax, reduce LVPmin of the model animals andthere was significant difference when comparing with that of the modelgroup, P<0.05, while was less effect on the right ventricle.

Results of the experiment showed that rhNRG-1 β could effectively treatthe congestive heart failure caused by constriction of inferior venacava.

Example 7 Rhesus Long-Term Toxicity Study of Recombinant HumanNeuregulin-1 β S177-Q237 for Injection Abstract

Long-term toxicity experiment on intravenous injection of 7.5, 15, 75μg/d of Recombinant Human Neuregulin-1 β_(S177-Q237) for Injection torhesus (the dosage used was based on the effective dosage in micepharmacodynamic model and conversed to dosage equivalent to 2, 4, 20times that of the dosage for monkey; and based on dog pharmacodynamicmodel and conversed to dosage equivalent to 4, 5, 9, and 45 times thatof dosage for monkey), excipient was used as control; monitoring wascontinuously carried out for 3 weeks after drug withdrawal. Toxicreactions of Recombinant Human Neuregulin-1 β S177-Q237 for Injectionand their severity to organism was studied, to look for target organs ofthe toxic reactions and reversibility of the damage and to determinedosage that does not cause toxic reaction and to serve the dosage as areference for safe dosage to be used in human.

The experiment animals were randomly divided into 4 groups based ontheir body weight with 6 animals, 3 male and 3 female, in each group.Volume of the drug administered was 1 ml/kg body weight. Body weight wasmeasured weekly and dosage was regulated according to the body weightmeasured. The drug was administered every day in the morning forconsecutive 3 weeks. Electrocardiogram, hematological, biochemistry,urine and fecal testing and determination of antibody were carried outprior to the drug administration and 10 days and 21 days after thebeginning of drug administration respectively. Fundus examination wasperformed under anesthesia at 22 days and 42 days (counted from thefirst day of drug administration) respectively, then ⅔ and ⅓ animalswere killed, autopsied and studied pathohistologically, bone marrowsmear was prepared at the same time. Echocardiography was performedunder anesthesia one day prior to the autopsy.

No animal death associated with the testing drug happened during theexperiment. Vomiting, nausea and salivation occurred in part of theanimals in each drug taken group 1 week after the beginning of drugadministration; pilo-erection, fur without luster, reduction ofactivity, anorexia were seen 2 weeks after the drug administration; mildpaleness, hardening of regional skin and vessels was discovered at thesite of drug injection; the above-mentioned symptoms and signdisappeared or reduced 3 weeks after drug withdrawal. No abnormalmanifestations were seen in animals of the control group during periodof drug administration and convalescent period after drug withdrawal.

Food ingestion reduced significantly and body weight lowered markedly inthe high dosage group (self-comparison prior to and after the drugadministration showed that the difference was significant; whereas therewas no significant difference when comparing with that of the controlgroup). There were no significant changes of body temperature in all theanimals prior to and after the drug administration.

Hematological, biochemistry examination, urine and fecal testing showedno significant toxicological changes.

Electrocardiography: no significant deceleration of heart rate was seenin conscious animals at 10-day of drug administration, while significantreduction of heart rate happened at 3-week of drug administration inconscious animals of every drug taken group, probably due topharmacological effect of the testing drug; in addition, relatively highvoltage of R and S wave in V1 and V3 leads of electrocardiogram,probably associated with variation of chest leads of the animals; nosignificant abnormality in echocardiogram, heart rate and other indexand no hypertrophic changes of the myocardium were seen pathologically.

No abnormal changes in fundus examination were seen.

No significant toxic pathologic changes were seen in the bone marrowsmear.

Autopsy performed at 3-week of drug administration showed pericardialeffusion in 3 animals each in both medium and high dosage groups and 2-3ml of transudate could be drawn out in each of them; hydrocephalus inthe subarachnoid space was discovered in 2 animals of the high dosagegroup and 0.5 and 3 ml of transudate was drawn out from each of themrespectively.

Various degree of vacuole appeared in the cytoplasma of myocardium,vascular congestion and mild edema under the pia mater were seen inthose with hydrocephalus. All of them had something to do with thetesting drug.

Antibody testing showed negative results.

The gastrointestinal symptoms such as vomiting, nausea and anorexia ofthose animals and the resulting reduction of body weight in the highdosage group due probably to distribution and elimination of the testingdrug in the gastrointestinal tract. Symptoms such as paleness, hardeningof skin at the injection site, pericardial effusion and hydrocephalus,and without entirely recovery of hydrocephalus of high dosage groupanimals 3 weeks after drug withdrawal demonstrated that capillaryexudate and transudate syndrome in rheusus can be caused by medium andhigh dosage of Recombinant Human Neuregulin-1 β_(S177-Q237) forInjection. In addition, deceleration of heart rate in conscious animalsand vacuole in the cytoplasma of myocardium of high dosage group animalswere all associated with the testing drug.

Conclusion:

Daily intravenous injection of 7.5, 15, 75 μg/d of Recombinant HumanNeuregulin-1 β_(S177-Q237) for Injection to rhesus for a total of 3weeks and continuously monitoring for 3 weeks were carried out. Theresults showed that more than 10 days of consecutive injection ofRecombinant Human Neuregulin-1 β_(S177-Q237) for Injection could causedeceleration of heart rate in conscious animals; it may lead togastrointestinal reaction; caused pericardial effusion, hydrocephalusand mild congestion and edema under the pia mater in rhesus of mediumand high dosage group, the hydrocephalus did not recover entirely 3weeks after drug withdrawal in the high dosage group animals; palenessand hardening of skin and vessels in the injection site recoveredgradually after drug withdrawal. Those manifestations demonstrated thatthe possible cause was capillary transudate-exudation syndrome. Highdosage of Recombinant Human Neuregulin-1 β_(S177-Q237) for Injectioncould cause vacuole in the cytoplasma of myocardium; antibody testingshowed negative results. The dosage level that did not cause pericardialeffusion and hydrocephalus during 3-week's drug administration and3-week's convalescent period was 7.5 μg/kg/d.

1. Objectives of the Experiment

To study the toxic reactions of Recombinant Human Neuregulin-1β_(S177-Q237) for Injection and their severity to organism, to look fortarget organs of the toxic reaction and reversibility of the damage, todetermine dosage that does not cause toxic reactions and to serve thedosage as a reference for safe dosage to be used in human.

2. Testing Drug:

-   2.1 Name of the drug: Recombinant Human Neuregulin-1 β_(S177-Q237)    for Injection.-   2.2 Batch number: 200210024.-   2.3 Institute that provided the drug: Zensun (Shanghai) Science &    Technology Development Company Ltd, Address: 2^(nd) floor of C    building, 328 Bi Bo Road, Zhang Jiang High Tech Zoon.-   2.4 Content: 3.75 mg/ml.-   2.5 Specific activity: 1.12×10⁴ U/mg.-   2.6 Character: transparent colorless solution.-   2.7 Storage: Store at 4° C.-   2.8 Excipient: 0.15M NaCl, 10 mM sodium phosphate, pH 6.0.-   2.9 Preparing: Dilute with normal saline to the needed    concentration.

3. Animals:

-   3.1 Species of the animals: Rhesus.-   3.2 Source of animals: Da Li Ji Rhesus Breeding Farm, Li Xing    County, Anhui Province, certificate of competency: Wan Fa Xun Fan    No. 2002-6.-   3.3 Reception Date of the animals: 13^(rd) Oct. 2002.-   3.4 Body weight: 2.6-5.9 kg at the beginning of drug administration.-   3.5 Gender: Half male and half female.-   3.6 Number of animals: a total of 24 animals.-   3.7 Marks on the animals: Chest card was used to identify individual    animal.-   3.8 Feeding condition: One animal for each cage, fed with granule    feedstuff (provided by Shanghai Shi Ling Science & Technology    Company Ltd). The animals were fed with 100 gram feedstuff two times    daily in addition to about 100 gram of fruit. The room temperature    was kept at 20-25° C., relative humidity of 50%-70%, illumination    for 12 hours every day.-   3.9 Environmental adaptation time: Breeding for 25 days for    environmental adaptation.

4. Dosage: 4.1 Dosage Set Up:

-   -   Control group: 0 μg/kg/d (injected with equal volume of        excipient);    -   Low dosage group: 7.5 μg/kg/d (comparable to 2 times that of        monkey equivalent dosage);    -   Medium dosage group: 15 μg/kg/d (comparable to 4 times that of        monkey equivalent dosage);    -   High dosage group: 75 μg/kg/d ((comparable to 20 times that of        monkey equivalent dosage).

4.2 Rationale of the Dosage Set Up:

-   -   The scheduled indication for Recombinant Human Neuregulin-1        β_(S177-Q237) for Injection was for therapy of heart failure.        The mice pharmacodynamic model was pro-cardiac Coxsacki B3 virus        induced myocarditis model, dosage used were 7.5, 15 and 30        μg/kg/d (equivalent to 1.875, 3.75 and 7.5 μg/kg/d used for        monkey), injected intravenously for consecutive 5 days, the        pathologic score was 1.5, 0.7 and 0.56 respectively (the        criteria for pathologic scoring is: score 0: area of lesions=0%,        score 1: area of lesions=25%, score 2: area of lesions=50%,        score 3: area of lesions=100%). Therefore, dosage of 15 and 30        μg/kg/d could significantly reduce damage to the heart. The        scheduled route of drug administration was intravenous        injection, once every day for consecutive 3-5 days. The MTD of        acute toxicity experiment on intravenous injection of        Recombinant Human Neuregulin-1 β_(S177-Q237) for Injection was        35 mg/kg. Take equivalent dosage for monkey of 3.75 μg/kg/d as        significantly effective dosage, the long-term toxic dosage for        animals will be tentatively set at 2, 4, and 20 times that of        the animals effective dosage.

5. Course of Drug Administration:

-   -   Once every day for consecutive 3 weeks.

6. Convalescence Period:

-   -   3 weeks.

7. Administrated Drug Volume:

-   -   1.0 ml/kg body weight.

8. Route of Drug Administration:

-   -   Slow intravenous injection, the same as that used clinically.

9. Method of the Experiment:

Albendazole was used to kill intestinal parasites and tuberculin testwas performed prior to buy in the experiment animal, then environmentaladaptation; hematological, biochemistry, urine testing andelectrocardiography were performed two times to ensure that theexperiment animals were in healthy condition. 4 groups were dividedbased on body weight and gender of the animals, with 6 animals in eachgroup, half male and half female. The volume of drug administered was1.0 ml/kg body weight, body weight was measured weekly prior to eachdrug administration and the volume of drug to be administered will beadjusted according to the body weight measured, ⅔ and ⅓ of the animals(2 female and 1 male monkeys each) in each group were killed at 24 hoursafter 3-week of drug administration and at the end of 3-week after theconvalescence period, autopsy performed, organs weighed, organcoefficient calculated and pathohistological examination carried out.Blood was withdrawn for hematological and biochemistry examination andbone marrow smear was performed at 10-day of drug administration andprior to autopsy.

9.1 Reagent:

-   -   9.1.1 Reagent for hematological testing.    -   9.1.2 Reagent for serum biochemistry testing: Trace imported        biochemistry reagent.    -   9.1.3 Main reagent for antibody detection.        -   {circle around (1)} Coating liquid: NaHCO₃ 0.293 g, Na₂CO₃            0.159 g, dissolve in 100 ml water.        -   {circle around (2)} Substrate buffer solution: pH 5.0,            citric acid 1.02 g, Na₂HPO₄.12H₂O 3.68 g dissolve in water.        -   {circle around (3)} Cleaning solution: 0.01M PBS adds 1:2000            Tween-20        -   {circle around (4)} Sealing solution: evaporated skimmed            milk 5 g, dissolve in 100 ml, pH 7.4 0.01M PBS.        -   {circle around (5)} HRP labeled rat anti-monkey second            antibody: Product of Sigma Company of the U.S. batch number:            A-2054.        -   {circle around (6)} Tween-20: Separated package of imported            raw material, provided by Zensun (Shanghai) Science &            Technology Company Ltd.        -   {circle around (7)} Tetramethylo-aminobenzene (TMB:            Separated package of imported raw material, provided by            Shanghai Huamei Bioengineering Company.        -   {circle around (8)} H₂SO₄: analytical reagent, Shanghai Ling            Feng Chemical Reagent Co Ltd.

9.2 Equipment:

-   -   Roche Hematology Vet Blood Cell Counter.    -   Hitachi-7060 Automatic Biochemistry Analytic Machine.    -   550 Enzyme labeling machine: product of BIO-RAD of the U.S.    -   Heraeus Low temperature centrifuge, Heraeus Company of Germany.    -   Hewlett Packard sonos 5500 echocardiography machine, with S4        probe.

9.3 Hematological and Serum Biochemistry Testing Methods: Refer to theFollowing Table

Method of Hematological Testing

Topic of testing Method of testing WBC white blood cell InstrumentalAnalysis RBC red blood cell Instrumental Analysis PLT plateletInstrumental Analysis Ht hematocrit Instrumental Analysis Hb hemoglobinInstrumental Analysis MCV mean corpusclular volume Instrumental AnalysisMCH mean corpusclular hemoglobin Instrumental Analysis MCHC meancorpusclular hemoglobin Instrumental Analysis concentration Retreticulocyte count BrilliantCresyl blue method DC differentiation countWright stain CT clotting time Slide method

Serum Biochemistry Testing Method

Testing Method ALT/GPT alanine aminotransferase IFCC w/o P-5-P AST/GOTaspartate aminotransferase IFCC w/o P-5-P ALP alkaline phosphataseTris/Carb LDH lactic acid dehydrogenase L→P enzyme method CPK creatinephosphokinase NAC enzyme method BUN blood urea nitrogenUrease-GLDH-Kinetic CRE creatinine Jaffe-Kinetic GLU glucose OxidaseT-Bil total bilirubin Dimethyl sulfoxide method T-CHO total cholesterolEnzymatic TP total protein Biuret ALB albumin Bromcresol green method K⁺kalium Electrode method Na⁺ natrium Electrode method Cl⁻ chlorideElectrode method P⁺⁺⁺/PHOS inorganic phosphorus Phosphomolybdate-UV Ca⁺⁺calcium Orthocresol phthalein complexon method Mg⁺⁺ magnesium Calmagitecomplexometric indicator

9.4 Method of Antibody Detection

Coating antigen: Recombinant Human Neuregulin-1 β_(S177-Q237) forInjection was diluted to 6 μg/ml with coating buffer solution, add into96 hole enzyme labeled plate with 100 μl/hole, 37° C. for 1 hour.

Sealing: Wash the plate 5 times, prepare 5% evaporated skimmed milk withcleaning solution.

Dilute the scheduled for testing serum; dilute the sample with sampledilution solution, dilute gradient is 1:100.

Add sample: Wash the sealed enzyme labeled plate for 3 times, addscheduled for testing serum, 100 μl/hole, 37° C. for 1 hour.

Add enzyme labeled antibody: Wash the plate 5 times, add 1:1000 dilutedHRP labeled rat anti-monkey immunoglobulin, 1041/hole, 37° C. for 1hour.

Substrate: Wash the plate 5 times, add newly prepared substrateoperating fluid, 100 μl/hole, 37° C. for 10 minutes.

Ending: add 2N H₂SO₄, 50 μl/hole to end the reaction.

Odd value detection: Positive result was defined as OD value of testingsample was 2.1 time greater than that of the negative control (serum was1:100 diluted).

10. Observation and Research Period 10.1 Death Status:

-   -   Monitoring was carried out once or twice daily and the time of        animal death will be recorded if there is any animal death.

10.2 General Symptoms:

-   -   Including general appearance, sign, behavior activity, blood or        exudation attached on the cage surface, luster of the fur. The        observation was carried out once or twice daily.

10.3 Body Weight:

-   -   Body weight was measured once daily prior to drug        administration.

10.4 Body Temperature:

-   -   Body temperature was taken prior to and 1 hour after the drug        administration at 1, 3 and 5-day of drug administration.

10.5 Feedstuff Ingestion:

-   -   Granule feedstuff 200 g was given to each monkey daily, with 100        g of fruits, calculate the consumption of food per animal per        day.

10.6 Electrocardiograph:

-   -   2 electrocardiograph examinations was performed prior to the        drug administration and also at 10 days after the beginning of        drug administration, at the end of drug administration and        during the convalescence period respectively, P-R, QRS, QT and        ST value were calculated.

10.7 Echocardiograph:

-   -   After ending the drug administration and 1 day prior to autopsy        at the end of convalescence period, the animals scheduled to be        autopsied will be anesthetized with intravenous injection of 3%        pentobarbital (30 mg/kg), then echocardiography performed to        determine the intraventricular thickness (IVS), postereior wall        of left ventricule (PW), left ventriclular end diastolic volume        (LVDd), left ventricular end systolic volume (LVDs), ejection        fraction (EF), shortening fraction (Fs), mitral valve blood flow        peak value (MV), aortic valve blood flow peak value (AV) and        heart rate (HR).

10.8 Hematological Index:

-   -   Two times prior to the drug administration, 10-day after the        beginning of the drug administration, at the end of drug        administration and at the end of 3-week's convalescence period        prior to autopsy, 0.5 ml blood was drawn from the saphenous        veins, treated with 3.8% EDTA for anticoagulation, testing for        the following index: red blood cell count (RBC), reticulocyte        count (Ret), hemoglobin (Hb), white blood bell count (WBC) and        differentiation count including: neutrophil (N), eosinophil (E),        Lymphocyte (L) and monocyte (M), platelet count (PLT), clotting        time (CT), hematocrit (Ht), mean corpuscular volume (MCV), mean        concentration hemoglobin (MCH) and mean corpuscular hemoglobin        concentration (MCHC).

10.9 Serum Biochemistry Index:

-   -   Two times prior to the drug administration, 10-day after the        beginning of drug administration, at the end of drug        administration and 3-week after convalescence period prior to        autopsy, 5 ml blood was drawn from the saphenous veins, serum        was separated after centrifugation and the following items were        tested: aspartate aminotransferase (AST), alanine        aminotransferase (ALT), alkaline phosphatase (ALP), lactic acid        dehydrogenase (LDH), creatine phosphokinase (CPK), blood urea        nitrogen (BUN), total protein (TP), albumin (ALB) glucose (GLU),        serum total bilirubin (T-Bil), creatinine (CRE), total        cholesteral (T-CHO), natrium (Na⁺), kalium (K+), chloride (Cl⁻),        calcium (Ca⁺⁺), magnesium (Mg+⁺) and phosphorus (P⁺⁺⁺).

10.10 Urine Testing

-   -   Two times prior to drug administration, 10-day after the        beginning of drug administration, at the end of drug        administration, 3-week after convalescence period prior to        autopsy, urine sample was collected for testing white blood        cell, nitrite, pH value, urine protein, glucose, ketone body,        urobilinogen, urine bilirubin and hemoglobin.

10.11 Fecal Testing

-   -   Fecal parasitic ovum and occult blood were tested prior to drug        administration. Fecal occult blood test was also performed at        10-day after the beginning of drug administration, at the end of        drug administration and 3-week after convalescence period.

10.12 Fundus Examination:

-   -   Fundus examination was carried out under anesthesia prior to        autopsy.

10.13 Systemic Autopsy:

-   -   ⅔ and ⅓ animals in each group (with 2 female and 1 male animal        each) were killed 24 hours after 3-week of drug administration        and at the end of convalescence period after the end of drug        administration, and autopsy carried.

10.14 Organ Coefficient:

-   -   Heart, brain, liver, spleen, lungs, kidneys, adrenals, thymus,        lymphnodes, thyroid, testis or uterus, ovary or prostate were        taken out, their weight measured and the coefficient calculated.

10.15 Pathohistological Examination:

-   -   Pathohistological examination of the following organs were        carried out: heart (anterior wall of left ventricle, right        ventricle, interseptum, left atrium, right atrium), liver,        spleen, lungs, brain, stomach, duodenum, ileum, colon, kidneys,        urinary bladder, adrenals, pituitary, thyroid, thymus, pancreas,        testis, prostate, ovary, uterus, lymph nodes (cervical,        mesentery), vessels and subcutaneous tissue of the injected        site.

10.16 Bone Marrow Examination:

-   -   Bone marrow sample from the femoral bone was taken out prior to        autopsy under anesthesia, smear, staining and optic microscopic        examination carried out to study the magakaryocyte system,        granulocyte and erythrocyte system, lymphocyte, plasmocyte and        other type of cells. 5×100 nucleated cells in the four quadrants        and central part will be counted, GE ratio calculated and        photographs taken.

10.17 Serum Recombinant Human Neuregulin-1 β_(S177-Q237) for InjectionAntibody Detection:

-   -   1, 2 and 3-week after the beginning of drug administration, and        at the end of convalescence period serum Recombinant Human        Neuregulin-1 β_(S177-Q237) for Injection antibody was detected,        and drug administration will be stopped whenever antibody was        detected and the detected antibody being neutralizing antibody.

10.18 Data Processing and Statistic Analysis:

-   -   Variance analysis was carried as statistical testing for data        collected from various dosage groups and control group.

11. Results of the Experiment: 11.1 Death Status:

No animal death associated with the testing drug happened during theexperiment period. At the end of 3-week of drug administration,significant deceleration of heart rate (20-30 beat/min) of 1 animal each(5# and 6#) in the control group and low-dosage group were seen throughechocardiography examination under anesthesia at the end of 3-week'sconvalescence period; being manifestation of over-anesthetization, thus,autopsy was carried out one day before scheduled. (Table 1).

11.2 General Manifestation:

Vomiting happened in 1 male monkey in high-dosage group 30 minutes afterintravenous injection at 2-day of drug administration. During the 2-7days of drug administration, 1 animal each in the medium and high-dosagegroup vomited in 20-30 minutes after intravenous injection. Salivationhappened in part of the animals in every group after 1-week of drugadministration, the rate of occurrence was significantly higher inhigh-dosage group than that in other drug taken groups; only 1 animal inlow-dosage group showed salivation at 9-day of drug administration.Pilo-errection, lost of luster of fur, reduced activity, anorexiaappeared in high-dosage group at 2-week of drug administration. Mildpaleness, hardening of regional skin and vessels in the injection sitebegan from the 3^(rd) day of drug administration, exaggeratedprogressively and was dosage dependent. The aforementioned symptoms andsigns disappeared or improved at 3-week of convalescence period afterdrug withdrawal. Light yellow color stool diarrhea and withoutregularity happened in animal 21# of high-dosage group. No abnormalmanifestation appeared in animals of the control group during period ofdrug administration and convalescence period after drug withdrawal.

11.3 Changes of Body Weight:

No significant difference in body weight (P>0.05, Table 2; FIG. 1) wasseen between animals of the control group and various drug taken groups;self-comparison in animals prior to and after drug administration showedsignificant reduction of body weight in animals of high-dosage group(P<0.01).

11.4 Body Temperature:

No significant difference in body temperature taken prior to drugadministration and 1 hour after drug administration at 1, 3 and 5-day ofdrug administration was seen (Table 3).

11.5 Feedstuff Ingestion:

Significant remnant of feedstuff and various amount of ingestion from 50to 150 g of feedstuff was seen in animals of medium and high-dosagegroups after 1-week of drug administration. No remnant of feedstuff wasseen in animals of the control group and low-dosage group and no remnantof feedstuff happened in all animals during convalescent period.

11.6 Results of Electrocardiograph Examination:

No significant deceleration of heart rate was seen in all the consciousanimals of every drug-taken group at 10-day of drug administration(P>0.05 when comparing with that of the control group), and significantdeceleration of heart rate happened in conscious animals of every drugtaken group, and P<0.05, P<0.01, when comparing with that of the controlgroup; at the same time, P-R, QRS and QT interval were prolongedaccordingly, being more marked in the low and medium dosage groups, theyreturned to normal 3-week after drug withdrawal; Voltage of Rv1 inanimals of medium and high-dosage group was higher, while Sv1 in high,low and medium dosage group were deeper and voltage of Rv3 in every drugtaken groups were higher. (P<0.05; P<0.01 and P<0.001, Table 4-7). Therewas mild fluctuation in other individual parameter collected indifferent time, however, all were within normal range.

11.7 Results of Echocardiograph:

No significant difference in parameters of echocardiogram in controlgroup and various drug taken group at 3-week of drug administration andat the end of 3-week of convalescent period were seen (P<0.05, Table8-9).

11.8 Results of Hematological Examination:

No significant abnormal changes in addition to fluctuation within normalrange of individual parameters during the entire experiment period wereseen (Table 10-13).

11.9 Results of Serum Biochemistry Testing:

No significant abnormal changes in addition to fluctuation within normalrange of individual parameters during the entire experiment period wereseen (Table 14-17).

11.10 Results of Urine Testing:

Increase of urine protein, positive ketone body, but without regularity,in urine testing of individual animals in every drug taken group andcontrol group were seen prior to drug administration, at 10-day and3-week after the beginning of drug administration, and at 3-week ofconvalescence period prior to autopsy. Transient increase of red bloodcells in urine was seen in individual animals of control group and everydrug taken group. No other significant abnormality was seen (Table18-21).

11.11 Results of Fecal Testing:

Negative Fecal occult blood test and no fecal parasitic ovum were seenin all the animals prior to drug administration, at 10-day and 3-weekafter the beginning of drug administration, and at 3-week ofconvalescence period prior to autopsy (Table 22).

11.12 Results of Fundus Examination:

No abnormality was seen in fundus examination performed at 3-week ofdrug administration and at 3-week of convalescence period prior toautopsy (Table 23).

11.13 Systemic Anatomy:

Small amount of effusion in the pericardial cavity of 2# male monkey and23#, 24# female monkey in medium dosage group were discovered and 2-3 mlof clear slight yellow color fluid was drawn out in each animal at3-week of drug administration; hydrorcephalus in the subarachnoid spacewas seen in 1# and 21# animal of the high-dosage group, and 1-2 ml ofclear slight yellow color fluid was drawn out from each animal; there iseffusion in peritoneal cavity, bleeding spot on the surface of colonicmucosa of 21# animal together with focus of ulcer in 21# animal.Congestion and hemorrhagic lesion in pulmonary lobe was seen in 22# malemonkey of high-dosage group. 814 monkey of the control group dies priorto autopsy due to over-anesthetization, significant congestion andpatchy bleeding in the lungs were seen during autopsy, no othersignificant abnormality was seen.

At the end of 3-week's convalescence period, hydrocephalus in thesubarachnoid space was seen in one male and one female animals (10#,14#) of high-dosage group, 0.5 ml and 3 ml fluid were drawn out fromthem respectively; there were no significant abnormality seen in animalthat was over-anesthetized and autopsied in advance.

No abnormality was seen in other organs.

Results of testing for the afore-mentioned effusion demonstrated thatthey were all transudate.

11.14 Weight of the Organs and their Coefficient

Weight of major organs were measured and organ coefficient calculatedduring the autopsy of all animals. No abnormal changes in organ weight,which has something to do with the testing drug, were seen (Table24-25).

11.15 Results of Pathohistological Examination:

Light stained vacuole appeared in cytoplasma of myocardium of the atriumand ventrircle of 1#, 11# male monkey and 21# female monkey ofhigh-dosage group, with their severity of “+” or “+ to ++”, striatedstructure was maintained; there was diffusive vacuole-like degeneration,without significant abnormality in endocardium and pericardium. On theother hand, vascular congestion and mild edema were seen in thesubarchnoid space of 11# and 21# monkey; there were inflammatory cellsinfiltration in part of the colonic mucosa of 21# animal; thickening ofavleola septum, congestion, patchy hemorrhage of the lung, full withedematous fluid in part of the alveola, partly tissue autocytolysis inkidneys, stomach, colon, intestine and pancreas were seen during autopsyof 8# animals that died before the autopsy. No other abnormality wasseen.

Micro-vacuole in the myocardial cytoplasma, vascular congestion in thesubarchnoid space was seen in 14# female monkey of the high-dosagegroup. No significant abnormality was discovered in animals autopsied inadvance due to over-anesthetization. No significant reaction to stimulusin addition to hemorrhagic changes in vessels within the injection sitein all the drug administration groups were seen. Sporadic, accidentallesions, such as inflammatory reaction in the lungs and gastrointestinaltract were seen in the remaining animals, most of them were spontaneouslesions (Table 26, 27 and Photos 1-22).

11.16 Results of Bone Marrow Examination:

Bone marrow was aspirated from the right hip bone prior to autopsy underanesthesia at 3-week of drug administration and 3^(rd) week ofconvalescence period, smear made, stained and megakaryocyte system,granulocyte system, erythrocyte system, lymphocyte and plasmocyte andother type of cells were examination under optic microscope. 5×100nucleated cells in the four quadrants and central part were counted anddifferentiation counting performed, GE ratio calculated andmicrophotography carried out.

Results: Normal proliferation in all the granulocyte, erythrocyte andmegakaryocyte system were seen, with normal ratio of granulocyte toerythrocyte system; no abnormal pathologic cells and no bone marrowtoxic pathologic damage was caused. (Table 28-29; photos 23-34).

11.17 Results of Serum Recombinant Human Neuregulin-1 β_(S177-Q237) forInjection Antibody Detection:

All the serum Recombinant Human Neuregulin-1 β_(S177-Q237) for Injectionantibody detection in all drug taken group at 1, 2, 3-week after thebeginning of drug administration and at the end of 3-week ofconvalescent period showed negative results. (Table 30).

12. Discussion:

Intravenous injection of 7.5, 15, 75 μg/d of Recombinant HumanNeuregulin-1 β_(S177-Q237) for Injection to rhesus (the dosage used wasequivalent to 2, 4, 20 times that of the dosage for monkey); excipientwas used as control; monitoring was carried out for 3 weeks after drugwithdrawal.

No animal death associated with the testing drug happened during theexperiment. Vomiting, nausea and salivation occurred in part of theanimals in each drug-testing group 1 week after the beginning of drugadministration; pilo-erection, fur without luster, reduction ofactivity, anorexia were seen in animals of the high-dosage group 2-weekafter the drug administration; mild paleness, hardening of regional skinand vessels at the site of drug injection; the afore-mentioned symptomsand signs disappeared or reduced 3 weeks after drug withdrawal. Noabnormal manifestations were seen in animals of the control group duringperiod of drug administration and convalescent period after drugwithdrawal.

Food ingestion reduced significantly and body weight lowered markedly inthe high dosage group in self-comparison prior to and after the drugadministration. There were no significant changes of body temperature inall the animals prior to and after the drug administration.

Hematological, biochemistry examination, urine and fecal testing showedno significant toxicological changes.

Electrocardiography: no significant deceleration of heart rate was seenin conscious animals at 10 days of drug administration, whilesignificant reduction of heart rate happened at 3-week of drugadministration in conscious animals of every drug taken group, probablydue to pharmacological effect of the testing drug; in addition,relatively high voltage of R and S wave in V1 and V3 leads ofelectrocardiogram, probably associated with variation of chest lead ofthe animals were seen; no significant abnormality in echocardiogram,heart rate and other index were discovered; no hypertrophic changes ofmyocardium was seen pathologically.

No abnormal changes in fundus examination were seen.

No significant toxic pathologic changes were seen in bone marrow smear.

Autopsy performed at 3-week of drug administration showed pericardialeffusion in 3 animals, each in both medium and high dosage groups and2-3 ml of transudate could be drawn out; hydrocephalus in thesubarachnoid space was discovered in 2 animals of the high dosage groupand 1-3 ml of transudate was drawn out.

Various degree of vacuole appeared in the cytoplasma of myocardium,vascular congestion and mild edema under the pia mater were seen inthose with hydrocephalus. All of them had something to do with thetesting drug.

Antibody testing showed negative results.

The gastrointestinal symptoms such as vomiting, nausea and anorexia ofthose animals could lead further to reduction of body weight in the highdosage group due probably to distribution and elimination of the testingdrug in the gastrointestinal tract. Symptoms such as paleness, hardeningof skin at the injection site, pericardial effusion and hydrocephaluswere seen, and without entire recovery of hydrocephalus in high dosagegroup animals 3 weeks after drug withdrawal, demonstrating thatcapillary exudate and transudate syndrome in rhesus can happened inmedium and high dosage of Recombinant Human Neuregulin-1 β_(S177-Q237)for Injection groups. In addition, deceleration of heart rate inconscious animals and vacuole in the cytoplasma of myocardium of highdosage group animals were all associated with the testing drug.

Conclusion: Daily intravenous injection of 7.5, 15, 75 μg/d ofRecombinant Human Neuregulin-1 β_(S177-Q237) for Injection to rhesus fora total of 3 weeks and continuously monitoring for 3 weeks were carriedout. The results showed that more than 10 days of consecutive injectionof Recombinant Human Neuregulin-1 β_(S177-Q237) for Injection couldcause deceleration of heart rate in conscious animals; it may lead togastrointestinal reaction, caused pericardial effusion, hydrocephalusand mild congestion and edema under the pia mater in rhesus in mediumand high dosage group animals, the hydrocephalus did not recoverentirely 3 weeks after drug withdrawal in the high dosage group animals;paleness and hardening of skin and vessels in the injection site andrecovered gradually after the drug withdrawal. Those manifestationsdemonstrated that the possible cause was capillary transudate-exudationsyndrome. High dosage of Recombinant Human Neuregulin-1 β_(S177-Q237)for Injection could cause vacuole in the cytoplasma of myocardium; allthe antibody testing showed negative results. The dosage level that didnot cause pericardial effusion and hydrocephalus during the 3-week'sdrug administration and 3-week's convalescent period was 7.5 μg/kg/d.

Example 8 In Vitro Determination of NGR-1 Activity (ELISA Test forKinase Receptor Activation) 1. Principle of the Experiment

HER2/neu gene encodes a trans-membrane protein p185, which is a tyrosineprotein kinase. Binding of Neuregulin-1 with ErbB3 or ErbB4 inducesheterodimer ErbB3-ErbB2 and ErbB4-ErbB2 formation and activates HER2encoded tyrosine protein kinase, mediating the transmission offunctioning signal of Neuregulin-1. Based on the fact that binding ofNeuregulin-1 with its receptors triggers phosphorylation of ErbB2protein, we establish a rapid, sensitive and high flux method for invitro quantitatively determining biological activity of RecombinantNeuregulin-1.

2. Experiment Material

-   2.1 96 holes cell cultural plate (Corning company); Costar 96 holes    ELISA detecting plate.-   2.2 Human breast cancer cell strain, introduced from the U.S. ATCC,    was cultivated in base cultural medium under 37° C. and 50% CO₂.-   2.3 Weighing a given amount of DMEM, quantifying to corresponding    volume, added 3.7 g/L of NaHCO₂, 0.1 g/L glutamine and 5.5 g/L of    HEPES.-   2.4 Base culture medium    -   DMEM culture medium with 10% fetal calf serum and insulin 9        mg/L, stored at 4° C. 2.5 Sterilized PBS (0.01M, pH 7.4).-   2.6 0.5% pancreatic enzyme    -   Preparing with Ca²⁺ and Mg²⁺ free PBS.-   2.7 Anti-ErbB2 monoclonal antibody coating buffer solution, lotion.    -   Select mouse anti-human ErbB2 extra-cell functioning domain H4        monoclonal antibody with no cross reaction with ErbB3 and ErbB4.    -   Coating buffer solution; pH 9.6, 0.05M carbonate buffer        solution.    -   Lotion: 0.01 M PBS+0.05% Tween-20.-   2.8 Horse-radish peroxidase (HRP) labeled mouse anti-human    phosphorylated protease monoclonal antibody (anti-P-tyr-HRP)-   2.9 Substrate, substrate buffer solution    -   Substrate (TMB): 2 mg/ml TMB (prepare with absolute alcohol).    -   Substrate buffer: 0.2M citric acid+0.1M Na₂HPO₄ (pH5.0).    -   Operating substrate: substrate buffer solution 9 ml+TMB 1 ml+3%        H₂O₂ 10 ul (prepared as needed).-   2.10 Termination agent    -   2N H₂SO₄.-   2.11 Cell defragmentation solution    -   150 mM NaCl+50 mM Hepes+1% Triton-X 100+2 mM (sodium        orthovanadate)+0.01% (thimerosol). One tablet of mixed protease        inhibitor (Tabletten, Proteasen-Inhibitoren-Cocktail) is added        into every 25 ml prior to the operation.-   2.12 Standard material and sample expected for testing.

3 Experiment Procedure

-   -   Process 3.1 was carried out on the first day, that of 3.2˜3.3 on        the second day, that of 3.4˜3.12 was performed on the third day;        in which, process of 3.1 and 3.2 should be carried out under        sterilization condition.

3.1 Inoculation of Cells

-   -   MCF-7 cells were amplified to a given amount, washed with        sterilized PBS solution, then digested with 0.25% trypsinase.        After counting, the concentration of cells was regulated with        base culture medium. The cells were added into 96 holes cell        culture plate, 5×104/hole, 100 μl/hole, and cultured over night        in the culture box under 37° C. and 5% CO₂.

3.2 Cell Starvation

-   -   Suck up all the culture medium in the 96 holes plate, wash each        hole with 37° C. warmed PBS, then add 100 μl DMEM culture medium        (calf serum free and without insulin). Cells were cultured for        24 hours in the culture box under 37° C. and 5% CO₂.

3.3 Coating

-   -   Dilute the anit-ErbB2 extra-cell functioning domain H4 antibody        with coating buffer solution to be 6 μg/ml, then add 50 μl per        hole to the 96 holes ELISA plate, set over night (16-18 hours)        under 4° C.

3.4 Dilute Control Solution and Sample Solution Expected to be Tested

Dilute control solution and sample expected to be tested with DMEMculture medium respectively (calf serum free and without insulin) to be2 μg/ml, then again carry out 3 times gradient dilution with a total of9 dilution.

3.5 Phosphorylation of the Cells

Suck up the post-starvation 96 holes cell culture medium, add standardmaterial and sample expected to be tested, 100 μl per hole, set up 2double hole for each concentration. Set up negative control at the sametune (i.e. DMEM culture medium placebo control). Reaction for 20 minutesunder 37° C.

3.6 Decomposition of the Cells

Rapidly suck out the sample and wash once with PBS, 100 μl offragmentation solution was added into each hole, fragmenting for 30minutes in 4° C. refrigerator. Horizontally agitate under ice-bathcondition till all the anchorage-dependent cell drop down, 4° C., 15,000rpm centrifuge for 15 minutes.

3.7 Sealing the ELISA Detecting Plate

Wash the plate 5 times. Prepare 5% skimmed milk with wash solution, add200 μl to each hole of the plate, set under 37° C. for 2 hours.

3.8 Add Sample

After wash 3 times the sealed ELISA plate, add standard cellfragmentation solution and testing sample fragmentation solution with 90μl per hole, set up negative control at the same time, set for 1 hourunder 37° C.,

3.9 Add Enzyme Labeled Antibody

Wash the plate 5 times, dilute HRP enzyme linked mouseanti-phosphorylated tyrosine protein antibody with 1:500 lotion(determined by the product using guide and the using time), add 100 μlinto each hole of the plate. Set for 1 hour under 37° C.

3.10 Color Development of the Substrate

Wash the plate 5 times, prepared substrate working solution was addedinto with 100 μl per hole, set for 10 minutes under 37° C.

3.11 Termination

2N H₂SO₄ was added into with 50 μl per hole to terminate the reaction.

3.12 OD Value Reading

Colorimetric analysis on the ELISA reader, determine wave length of 450nm, reference wave length of 655, record the results.

4 Calculation

Construction with concentration of Recombinant Human Neuregulin-1 versusOD value and analysis was carried out with linear regression method,calculate the half effective dosage of each sample expected for testing.

5 Needed Reagent Formula 1. DMEM Base Culture Medium

Fetal calf serum 100 ml Insulin  9.2 mg

-   -   add into IL of DMEM culture medium, mixed well.

2. Cell Fragmentation Solution

NaCl 4.38 g HEPES 5.96 g sodium orthovanadate 0.368 g thimerosol 0.05 gTriton-X 100 5 mL

-   -   Dissolve into 500 mL H₂O

3. Coating Solution (pH 9.6)

NaHCO₃ 0.293 g Na₂CO₃ 0.159 g

-   -   Dissolve into 100 mL H₂O

4. 100 mL Substrate Buffer Solution (pH 5.0)

Citric acid 1.02 g Na₂HPO₄•12H₂O 3.68 g

-   -   Dissolve into H₂O

5. Sealing Solution (5% Skimmed Milk)

Skimmed milk powder    5 g Dissolve into 100 mL pH 7.4 0.01M PBS 6.0.01M PBS (pH 7.4) Na₂HPO₄•12H₂O 2.9014 g NaH₂PO₄•4H₂O 0.2964 g NaCl  8.5 g

-   -   Dissolve into 1000 mL H₂O

7. 0.01M PBS-T (PH 7.4)

-   -   Add 1 ml Tween 20 into 2000 mL 0.01M PBS, mixed well.    -   8. 2 mg/ml TMB

TMB 20 mg

-   -   Dissolve into 10 mL absolute alcohol

9. 20×PBS (1000 ML)

Na₂HPO₄•12H2O  58 g NaH₂PO₄•4H2O  5.9 g NaCl 170 g

-   -   Dissolve into 1000 mL H₂O.

The above examples are included for illustrative purposes only and arenot intended to limit the scope of the invention. Many variations tothose described above are possible. Since modifications and variationsto the examples described above will be apparent to those of skill inthis art, it is intended that this invention be limited only by thescope of the appended claims.

1. A combination, which combination comprises an effective amount of aneuregulin protein, or a functional fragment thereof, or a nucleic acidencoding a neuregulin protein, or a functional fragment thereof, or anagent that enhances production and/or function of said neuregulin, andan effective amount of a prophylactic or therapeutic agent for viralmyocarditis or dilated (congestive) cardiomyopathy (DCM).
 2. Thecombination of claim 1, wherein the neuregulin carries out itsanti-viral myocarditis or anti-DCM activity via binding with ErbB2-ErbB4receptors.
 3. The combination of claim 1, wherein the neuregulin isselected from the group consisting of neuregulin 1, neuregulin 2,neuregulin 3 and neuregulin
 4. 4. The combination of claim 3, whereinthe neuregulin 1 is neuregulin α2 or neuregulin β2.
 5. The combinationof claim 1, wherein the neuregulin fragment is a neuregulin β2 fragmentcomprising an amino acid sequence set forth in SEQ ID NO:4.
 6. Thecombination of claim 1, wherein the prophylactic or therapeutic agentfor viral myocarditis is selected from the group consisting of anantibiotic, a heart protective agent, an antioxidant and a nutrient formyocardium. 7-10. (canceled)
 11. The combination of claim 1, wherein theprophylactic or therapeutic agent for DCM is selected from the groupconsisting of a cardiac tonic, a diuretic, an angiotensin I-convertingenzyme inhibitor (ACEI), a calcium antagonist and a β-receptorantagonist. 12-17. (canceled)
 18. A method for preventing, treating ordelaying viral myocarditis or dilated (congestive) cardiomyopathy (DCM)in a mammal, which method comprises administering to a mammal, to whichsuch prevention, treatment or delay is needed or desirable, an effectiveamount of a neuregulin protein, or a functional fragment thereof, or anucleic acid encoding a neuregulin protein, or a functional fragmentthereof, or an agent that enhances production and/or function of saidneuregulin, whereby said viral myocarditis or DCM is prevented, treatedor delayed.
 19. The method of claim 18, wherein the neuregulin carriesout its anti-viral myocarditis or anti-DCM activity via binding withErbB2-ErbB4 receptors.
 20. The method of claim 18, wherein theneuregulin is selected from the group consisting of neuregulin 1,neuregulin 2, neuregulin 3 and neuregulin
 4. 21. The method of claim 20,wherein the neuregulin 1 is neuregulin α2 or neuregulin β2.
 22. Themethod of claim 18, wherein the neuregulin fragment is a neuregulin β2fragment comprising an amino acid sequence set forth in SEQ ID NO:4. 23.The method of claim 18, wherein the mammal is a human.
 24. The method ofclaim 18, wherein the viral myocarditis is caused by or associated withinfection of a virus selected from the group consisting of CoxsackieGroup A virus, Coxsackie Group B virus, ECHO virus and polio virus. 25.(canceled)
 26. The method of claim 18, wherein the viral myocarditis iscomplicated by pericarditis or endocarditis.
 27. The method of claim 18,wherein the viral myocarditis has a clinical feature selected from thegroup consisting of myocardial damage, heart dysfunction, arrhythmia,systemic symptom and cardiomyopathy. 28-30. (canceled)
 31. The method ofclaim 18, wherein the DCM has a clinical feature selected from the groupconsisting of ventricular hypertrophy, myocardial pump function failureand congestive heart failure. 32-53. (canceled)
 54. A method forpreventing, treating or delaying cardiac toxicity in a mammal to whichsuch prevention, treatment or delay is needed or desirable, comprisingadministering to a mammal in vivo an effective amount of a prophylacticor a therapeutic agent and an effective amount of: (i) a neuregulinprotein or a functional fragment thereof; (ii) a nucleic acid encoding aneuregulin protein or a functional fragment thereof; or (iii) an agentthat enhances production or function of said neuregulin, whereby saidcardiac toxicity associated with administration of said prophylactic ortherapeutic agent is prevented, treated or delayed. 55-60. (canceled)61. The method of claim 54, wherein the cardiac toxicity to beprevented, treated or delayed comprises a decrease in left ventricularejection fraction (LVEF), a decrease in stroke volume (SV), a decreasein cardiac output (CO), or a decrease in cardiac index (CI). 62.-94.(canceled)
 95. A method for preventing, treating or delaying myocardialinfarction in a mammal, which method comprises administering to amammal, to which such prevention, treatment or delay is needed ordesirable, an effective amount of a neuregulin protein, or a functionalfragment thereof, or a nucleic acid encoding a neuregulin protein, or afunctional fragment thereof, or an agent that enhances production and/orfunction of said neuregulin, whereby said myocardial infarction isprevented, treated or delayed. 96-124. (canceled)